Method for isolation of unclipped HIV envelope protein

ABSTRACT

Methods for producing and isolating unclipped HIV env proteins are provided. According to the methods, an antibody directed to an HIV epitope spanning the env clip site is used to selectively separate unclipped HIV env protein from clipped env protein.

This is a divisional of application Ser. No. 08/101,669 filed on 2 Aug.1993, now abandoned, which application is a continuation of Ser. No.07/834,735 filed on 13 Feb. 1992 (abandoned), which application is adivisional of Ser. No. 07/504,785 filed on 3 Apr. 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to methods for the use of the HumanImmunodeficiency Virus, or HIV, envelope (env) polypeptides, especiallythe preparation and use of HIV env variants and antibodies to HIV env,in the vaccination and treatment of HIV-infected patients.

BACKGROUND OF THE INVENTION

Acquired immunodeficiency syndrome (AIDS) is caused by a retrovirusidentified as the human immunodeficiency virus (HIV). A number ofimmunologic abnormalities have been described in AIDS includingabnormalities in B-cell function, abnormal antibody response, defectivemonocyte cell function, impaired cytokine production, depressed naturalkiller and cytotoxic cell function, and defective ability of lymphocytesto recognize and respond to soluble antigens. Other immunologicabnormalities associated with AIDS have been reported. Among the moreimportant immunologic defects in patients with AIDS is the depletion ofthe T4 helper/inducer lymphocyte population.

In spite of the profound immunodeficiency observed in AIDS, themechanism(s) responsible for immunodeficiency are not clearlyunderstood. Several postulates exist. One accepted view is that defectsin immune responsiveness are due to selective infection of helper Tcells by HIV resulting in impairment of helper T-cell function andeventual depletion of cells necessary for a normal immune response. Invitro and in vivo studies showed that HIV can also infect monocyteswhich are known to play an essential role as accessory cells in theimmune response. HIV may also result in immunodeficiency by interferingwith normal cytokine production in an infected cell resulting insecondary immunodeficiency as for example, IL-1 and IL-2 deficiency. Anadditional means of HIV-induced immunodeficiency consists of theproduction of factors which are capable of suppressing the immuneresponse. None of these models resolves the question of whether acomponent of HIV per se, rather than infection by replicative virus, isresponsible for the immunologic abnormalities associated with AIDS.

The HIV env protein has been extensively described, and the amino acidand RNA sequences encoding HIV env from a number of HIV strains areknown (Modrow, S. et al., J. Virology 61(2):570 (1987). The HIV virionis covered by a membrane or envelope derived from the outer membrane ofhost cells. The membrane contains a population of envelope glycoproteins(gp 160) anchored in the membrane bilayer at their carboxyl terminalregion. Each glycoprotein contains two segments. The N-terminal segment,called gp120 by virtue of its relative molecular weight of about 120 kD,protrudes into the aqueous environment surrounding the virion. TheC-terminal segment, called gp41, spans the membrane. gp120 and gp 41 arecovalently linked by a peptide bond that is particularly susceptible toproteolytic cleavage, see e.g. McCune et al., EPO Application No. 0 335635, priority 28 Mar. 1988 and references cited therein.

Several approaches to an AIDS vaccine have been proposed, includinginactivated and attenuated virus vaccines, subunit vaccines fromvirus-infected cells, recombinantly produced viral antigens, vaccinesbased on synthetic peptides, anti-idiotypic vaccines, and viralcarrier-based vaccines, however no vaccination study published to datehas provided protection against challenge with virus. Several reviews ofHIV vaccine development have been published, e.g. Lasky, CriticalReviews in Immunology 9(3):153-172 (1989), Newmark, Nature 333:699 (23Jun. 23, 1988), and Fauci et al., Annals of Internal Medicine110(5):41-50 (1 Mar. 1989).

The use of whole (killed or attenuated) virus presents several problems,including the safety to workers producing the vaccine, and risk to thoseinoculated from incomplete inactivation of virus, or reversion of anattenuated virus to an active, virulent form. Both peptide and subunitvaccines could potentially have difficulty in obtaining their nativeconformations, and may only elicit humoral responses, perhaps noteliciting cell-mediated immunity. Another key difficulty in developingan AIDS vaccine lies in the variability of HIV from strain to strain, aswell as in the same strain over time.

Of the proteins encoded by the HIV genome, the molecules most frequentlyused for vaccine development are located on the surface of the virus.They mediate virus attachment and the spread of the virus bycell-to-cell fusion (syncytia formation) and are the vital proteins mostaccessible to immune attack. Currently, gp120 is considered to be thebest candidate for a subunit vaccine, because: (i) gp120 is known topossess the CD4 binding domain by which HIV attaches to its targetcells, (ii) HIV infectivity can be neutralized in vitro by antibodies togp 120, (iii) the majority of the in vitro neutralizing activity presentin the serum of HIV infected individuals can be removed with a gp120affinity column, and (iv) the gp120/gp41 complex appears to be essentialfor the transmission of HIV by cell-to-cell fusion.

Vaccination of animals of several species with recombinant vectors thatexpress HIV env are described in the literature. These vaccinationattempts elicited strain-specific humoral immune responses as well ascell-mediated responses (see e.g. Van Eendenburg et al., AIDS Researchand Human Retroviruses 5(1):41-50 (1989); Hu et at., Nature 320:537-540(1986); Chakrabarti et al., Nature 320:535-537 (1986); Zarling et al.,Nature 323:344-346 (1986); Hu et al., Nature 328:721-724 (1987); Zaguryet al., Nature 326:249-250 (1987); Zagury et al., Nature 322:728-731(1988).

Chimpanzees are the only nonhuman primate infectable with HIV andtherefore they are the closest-to-human animal model system forvaccine-challenge study. Despite the promise suggested by the immuneresponses discussed above, published vaccine studies have all failed toprotect chimpanzees from infection by HIV (see e.g. Hu et al., Nature328:721-724 (1987) (vaccinia virus-HIV env recombinant vaccine); Arthuret al., J. Virol. 63(12):5046-5053 (1989) (purified gp120); Berman etat., Proc. Natl. Acad. Sci. USA 85:5200-5204 (1988) (recombinantenvelope glycoprotein gp120); and Prince et al., Proc. Natl. Acad. Sci.USA 85:6944-6948 (1988) (purified human HIV immune globulin).

The Simian Immunodeficiency Virus (SIV) is a lentivirus which isindigenous to healthy African monkeys; SIV is the animal lentivirus mostclosely related to HIV. Letvin et al., Vaccines 87, Cold Spring HarborLab 209-213 (1987) discloses an unsuccessful attempt to immunize macaquemonkeys against SIV using an inactivated virus vaccine. Desrosiers etal., Proc. Natl. Acad. Sci. USA 86:6353-6357 (1989) reported protectionof two of six macaque monkeys against SIV by immunization with adetergent-disrupted whole virus SIV vaccine. Murphey-Corb et al.,Science 246:1293-1297 (1989) disclose protection of eight of nine rhesusmacaques against a SIV challenge by vaccination with aformalin-inactivated SIV whole virus vaccine.

It is therefore an object of this invention to provide vaccines capableof eliciting a protective immune response against HIV infection.

It is a further object of this invention to provide methods forpreparing such HIV vaccines, and appropriate immunization schedules forthe prevention and treatment of AIDS.

Other objects, features, and characteristics of the present inventionwill become apparent upon consideration of the following description andthe appended claims.

SUMMARY OF THE INVENTION

The objects of this invention are accomplished by the preparation andadministration an HIV antigen preparation which is suitable foradministration to a human or non-human primate patient having or at riskof having HIV infection, in an amount and according to an immunizationschedule sufficient to induce a protective immune response against HIV.

The vaccines of this invention may be administered alone or incombination with other HIV antigens, and in one or several immunizationdoses. Preferred immunization schedules are described, which generallyprovide for infrequent immunizations spaced at relatively longintervals, particularly a series of three or more inoculationsadministered over a period of one to two or more years.

This invention is particularly directed to vaccines comprising the HIVenv polypeptides gp120 and/or gp 160 which have a proteolytic clip sitebut have not been proteolytically cleaved at that internal clip site.This clip site of HIV env is believed to be located between amino acidresidues 315-316 of the gp120 of the HIV strain described in EP187,041A, commonly known as IIIB, or the equivalent region of other HIVstrains.

HIV env preparations which are devoid of material containing theinternal clip site are useful in vaccines for immunization against HIVinfection. This unclipped HIV env polypeptide, including variantanalogues thereof, is also useful in diagnostic assays for HIVneutralizing antibody in patient samples.

Methods of preparation of unclipped gp120 and gp160 are provided. Insome embodiments, fermentation processes are provided, comprisingexpression of gp120 in mammalian cell culture, grown in media which hasreduced or absent fetal calf or bovine serum; these fermentation methodsencourage expression ad recovery of unclipped HIV env polypeptides.Methods of preparation of the vaccines of this invention are alsodisclosed.

Monoclonal antibodies are provided which are characterized by theiraffinity for ligand, epitope binding, and ability to a) block CD4/gp120binding, b) neutralize HIV virions, c) reduce reverse transcriptaseactivity in vitro, and d) inhibit syncitia formation. In particularembodiments, monoclonal antibodies are provided that are specific forthe region of gp120 which contains the internal clip site. Theseantibodies are useful as diagnostics for the presence of HIV infectionin a patient or patient sample, and for affinity purification ofunclipped gp120 or gp160. These antibodies are also useful in passivelyimmunizing patients infected with HIV.

Antibodies directed to epitopes which span this clip site have beendescribed in the literature; however, it should be noted that, due tothe variety and confusion among authors currently as to numberingsystems for HIV env sequences, not all antibodies described in theliterature as directed to regions including amino acids 315-316 willactually span the clip site defined herein (see e.g. Matsushita et al.,J. Virol. (62:2107-2114 (1988); EPO Application No. EP 339 504; Ruscheet al., Proc. Natl. Acacl. Sci. USA, 85:3198-3202 (1988); Looney et al.,Science 241:357-359 (1988);

In certain embodiments, antibodies are provided which are capable of atleast partially blocking the bonding of recombinant gp120 to the T4 cellsurface marker CD4. In other embodiments, antibody is provided which isat least partially capable of blocking syncytia formation betweenHIV-infected and uninfected cells and/or blocking reverse transcriptaseactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of gp 120 showing disulfides andglycosylation sites. Roman numerals label the five disulfide-bondeddomains. The five hypervarlable regions of Modrow et al., J. Virol.61:570-578 (1987) are enclosed in boxes and labelled V1-V5.Glycosylation sites containing high mannose-type and/or hybrid-typeoligosaccharide structures are indicated by a branching-Y symbol, andglycosylation sites containing complex-type oligosaccharide structuresare indicated by a V-shaped symbol. The clip site is designated by anarrow.

FIG. 2A and 2B show the amino acid sequences of (FIG. 2A) the mature HIVenv glycoprotein gp120 from the IIIB isolate of HIV-1, and (FIG. 2B) theN-terminal sequence portion of the recombinant fusion glycoproteins (9AAor CL44) from the herpes simplex virus protein gD1. Fusion sites betweenthe gD1 and gp120 segments in the 9AA and CL44 constructions are markedwith (*) and (**), respectively. The letter T refers to observed trypticcleavage of the gp 120 segment, and the peptides are orderedsequentially starting at the N-terminus of the molecule. Lower caseletters following the T number indicate other unexpected proteolyticcleavages. The letter H refers to the observed tryptic cleavage of theherpes simplex gD1 protein portion of CL44. Peptide T2' contains thefusion site in CL44. The cysteine residues of gp120 are shaded, andpotential N-linked glycosylation sites are indicated with a dot abovethe corresponding Asn residue.

FIGS. 3A and 3B depict the composition of rgp120 and sgp160 immunogensused in the vaccination described in Example 1.

FIG. 3A is a schematic representation of the HIV-1 glycoproteinprecursor, gp160, and the two variant gp160 derived proteins, rgp120,and sgp1 used in the present study. Both proteins differ at the aminoterminus from wild-type gp160. In the rgp120 protein, the signalsequence and 31 residues of gp160 have been replaced with the signalsequence and 25 amino acids from the mature N-terminus of herpes simplexvirus type 1 glycoprotein D (HSV-1 gD)³. In the sgp160 protein, thesignal sequence and 12 amino acids of gp160 have been replaced with thesignal sequence and 9 amino acids from the mature N-terminus of HSV-1gD. In addition, the normal gp120/gp41 proteolytic processing site hasbeen removed by a deletion spanning amino acid residues 502-511.

FIG. 3B depicts silver-stained SDS-PAGE gel of purified rgp120 andsgp160 under reducing and non-reducing conditions. Both recombinantglycoproteins were purified from growth conditioned cell culturesupernatants by immunoaffinity chromatography and gel permeationchromatography to greater than 99% purity for rgp120 and greater than95% purity for sgp160. Under non-reducing conditions the rgp120contained less than 5% dimer while the sgp160 was approximately 50%dimer and higher order oligomers. Both proteins are subject to anendoproteolytic cleavage between arginine residue 315 and alanineresidue 316 in the V3 region of the gp120 portion of each molecule. Inrgp120 this results in the formation of an N-terminal 75 kD fragment anda 50 kD C-terminal fragment, while in sgp160 the same cleavage resultsin two fragments both of approximately 75 kD. On SDS-PAGE the fragmentsare visible only under reducing conditions because of disulfide bondingbetween cysteine residues 296 and 331. The rgp120 preparation containedless than 5% of the cleaved form whereas approximately 40% of the sgp160was cleaved.

FIG. 4 shows the results of an immunoblot depicting the cross reactivityof the antibodies of this invention with other isolates of HIV-1 besidesIIIB.

FIG. 5 is an autoradiograph showing analysis of ten monoclonalantibodies to sgp160. It can be seen that three major bands werespecifically immunoprecipitated by one or more of the sera tested, a 140kD band corresponding to sgp160, a 110-120 kD band corresponding torgp120, and a 75 kD band which represents proteolytic breakdown productsof gp120 and sgp160.

FIGS. 6A and 6B show the production of antibodies to rgp120 and sgp160in animals immunized with candidate HIV-1 vaccines. Chimpanzees wereimmunized with 300 μg per dose of rgp160 (x-247 and x-261), rgp120 (x262and x-265) or herpes simplex virus glycoprotein D (x-246), at times 0, 4weeks, and 32 weeks (arrows). All immunogens were incorporated inaluminum hydroxide adjuvant containing 2 mg equivalents of Al⁺³ per mgof protein. Blood was taken at the time points indicated, and the serawere analyzed for antibodies to rgp120 or sgp160 in the assays describedfor the in each of the specific panel descriptions. All animals werechallenged by intravenous injection of 40 TCID₅₀ units of HIV-1 at 35weeks. The open and closed squares represent the rgp120 immunizedanimals, x262 and x265, respectively. The open and closed circlesrepresent the rsgp160 immunized animals, x247 and x261, respectively.The control animal, x246, is represented by the open triangles.

FIG. 6A illustrates detection of antibodies to rgp120 byimmunoprecipitation of ¹²⁵ I-labeled rgp120 in a liquid phaseradioimmunoprecipitation assay.

FIG. 6B shows detection of antibodies to HIV-1 proteins using acommercial (Genetic Systems) HIV-1 antibody assay kit (ELISA).Measurements were carried out according to the manufacturersinstructions.

FIG. 7 shows immunoblot analysis of sera from animals immunized withcandidate HIV-1 vaccines. Sera from chimpanzees immunized with sgp160(x-247, x-261), rgp120 (x-262, x-265) or HSV-I gD (x-246) were dilutedand incubated with commercial (Dupont) immunoblot strips. The stripswere incubated with alkaline phosphatase coupled goat anti-human IgG(Cappel) and developed with Phospharase substrate system obtained fromKirkegaard and Perry Laboratories. The data shown represent resultsobtained on the same day using the same lot of assay strips. Week 35represents the time of challenge. The positive control consisted ofserum from an HIV-1 infected individual.

FIGS. 8A & 8B show antibodies that neutralize HIV-1 infectivity in vitroand bind to the major type specific neutralizing determinant (MND). Openand closed squares represent sera obtained from the rgp120 immunizedanimals 262 and 265, respectively. The open and closed circles representsera obtained from the rsgp160 immunized animals, 247 and 261,respectively. The open triangle represent the control animal (x246).

FIG. 8A shows the in vitro neutralizing activity in sera fromchimpanzees immunized with rgp120 and sgp160 in a neutralization assaysimilar to that described by Robertson et al., J. Virol. Methods20:195-202 (1988). Diluted samples of serum were incubated with 100TCID₅₀ units of virus (IIIB isolate) for 60 minutes at 20° C. Themixture was transferred to cell culture plates containing 5×10⁴ MT4cells and incubated for 7 days. Virus lysis of infected cells wasdetected through the use of MTT as a vital stain. Neutralization assayswere carried out in duplicate. Variation between replicates was lessthan one dilution (twofold). Sere from HIV-1 infected and uninfectedchimpanzees were used as a positive and negative controls and gaveneutralizing titers of 1:640 and <1:10 respectively.

FIG. 8B shows results of an ELISA assay to determine the relativeconcentration of antibodies reactive with the MND, where a syntheticpeptide consisting of the sequence: NNTRKSIRKSIRIQRGPGRAFVTIGKIG andcorresponding to amino acid residues 301 to 324 of gp120 from the IIIBisolate, was coated onto microtiter dish at a concentration of 2 μg perml. After an overnight incubation at 4° C., the coated wells were washedwith phosphate buffered saline (PBS) containing 0.05% Tween 20 andtreated with a blocking buffer consisting of 0.8% bovine serum albuminin PBS. Each sample of chimpanzee sere serially diluted over a range of1:10--to 1:10,240 and 100 μl were incubated in the wells for 1.5 hr atroom temperature. The wells were then washed four times with PBScontaining 0.05% triton X-100. Labeled horseradish peroxidase conjugatedgoat-antihuman IgG was incubated for 1 hr and the plates washed fourtimes with PBS, 0.05% Triton X-100. Antibody binding was indicated by achange in the color after the substrate o-phenylenediaminedihydrochloride was added. The colorometric reaction was stopped by theaddition of 2.5M H₂ SO₄, and the absorbance at 492 nm was measured in aplate reading spectrophotometer.

DETAILED DESCRIPTION OF THE INVENTION

HIV env is defined herein as the envelope polypeptide of HumanImmunodeficiency Virus as described above, together with its amino acidsequence variants and derivatives produced by covalent modification ofHIV env or its variants in vitro, as discussed herein. As used herein,the term "HIV env" encompasses all forms of gp120 and/or 160, e.g.including fragments, fusions of gp160/120 or their fragments with otherpeptides, and variantly glycosylated or unglycosylated HIV env. The HIVenv of this invention is recovered free of active virus.

HIV env and its variants are conventionally prepared in recombinant cellculture. For example, see EP publication No. 187041. Henceforth, gp120prepared in recombinant cell culture is referred to as rgp120.Recombinant synthesis is preferred for reasons of safety and economy,but it is known to prepare peptides by chemical synthesis and to purifyHIV env from viral culture; such env preparations are included withinthe definition of HIV env herein.

Genes encoding HIV env are obtained from the genomic cDNA of an HIVstrain or from available subgenomic clones containing the gene encodingHIV env. Cell cultures encoding gp120 of this invention have beendeposited with the ATCC.

This invention is directed to the HIV env polypeptides gp120 or gp160,and preferably gp120, which have an internal cleavage site referred toherein as the "clip site" but which are not clipped at that site. Thesepolypeptides are referred to herein as "unclipped HIV env", or moreparticularly "unclipped gp120" or "unclipped gp160". The clip site is abasic or dibasic residue susceptible to proteolytic cleavage.

As used herein, the term "clip site" does not refer to the cleavage sitebridging gp120 and gp41, but instead refers to a proteolytic clip sitewhich is located in gp 120 of HTLV-IIIB between Arg-Ala or Arg-Valresidues, or between similarly situated residues in HIV strainscurrently common in the United States and in Africa, and in SIV strains.See Stephens et al., Nature 343:219 (1990).

As shown by the arrow in FIG. 1, the clip site is located between aminoacid residues 285-286 of the mature HIV-1 gp120 amino acid sequence, notcounting any signal sequence or other upstream regions. For gp120sequences which include the native HIV-IIIB N-terminal signal sequence,the clip site is found between residues 315-316; this numbering is usedthroughout this description to conveniently connote the residues atwhich the clip site is located, however it is understood that thisinvention is not limited to clip sites at those specific residuenumbers. The same nucleotide and amino acid residue numbers may not beapplicable in other strains where upstream deletions or insertionschange the length of the viral genome and HIV env, but the regionencoding this portion of gp120 is readily identified by reference to theteachings herein. Also, variant signal sequences (such as thoseresulting from a fusion with a fragmented or heterologous signalsequence as discussed below may lead to a slightly different numbering,however the location of the clip site is discerned for all embodimentsby reference to the location of the arrow indicated in FIG. 1.

Included within the scope of unclipped HIV env as that term is usedherein are HIV envs having the amino acid sequences set forth in FIG. 1or 2, deglycosylated or unglycosylated derivatives of unclipped HIV env,homologous amino acid sequence variants of the sequence of FIG. 1 or 2,and homologous in vitro-generated variants and derivatives of unclippedHIV env, provided that all such variations do not interfere with theclip site, and which variants are capable of exhibiting a biologicalactivity in common with the HIV env of FIG. 1 or FIG. 2.

Clipped or unclipped HIV env or HIV env-fragment biological activity isdefined as either 1) immunological cross-reactivity with at least oneepitope of clipped or unclipped HIV env, or 2) the possession of atleast one adhesive or effector function qualitatively in common withclipped or unclipped HIV env. Examples of the qualitative biologicalactivities of the HIV env include the ability of gp120 to bind to thevital receptor CD4, and the ability of gp120 to interact with gp41 toinduce fusion of the viral and host cell membranes.

Immunologically cross-reactive as used herein means that the candidatepolypeptide is capable of competitively inhibiting the qualitativebiological activity of clipped or unclipped HIV env having this activitywith polyclonal antisera raised against the known active analogue. Suchantisera are prepared in conventional fashion by injecting goats orrabbits, for example, subcutaneously with the known active analogue incomplete Freund's adjuvant, followed by booster intraperitoneal orsubcutaneous injection in incomplete Freunds.

It is currently preferred that the unclipped HIV env preparations ofthis invention be substantially free of clipped HIV env fragments. Bysubstantially free it is meant that the preparations should be greaterthan 50 percent, more preferably 60-70 percent, still more preferably 80percent, and most preferably at least 90 percent free of clipped HIV envfragments.

The gp120 molecule consists of a polypeptide core of 60,000 daltons;extensive modification by N-linked glycosylation increases the apparentmolecular weight of the molecule to 120,000 (Lasky et al., Science233:209-212 (1985)). The amino acid sequence of gp120 contains fiverelatively conserved domains interspersed with five hypervariabledomains (Modrow, supra). The hypervariable domains contain extensiveamino acid substitutions, insertions and deletions. Sequence variationsin these domains result in up to 25% overall sequence variabilitybetween gp120 molecules from the various viral isolates. Despite thisvariation, several structural and functional elements of gp120 arehighly conserved. Among these are the ability of gp120 to bind to theviral receptor CD4, the ability of gp120 to interact with gp41 to inducefusion of the viral and host cell membranes, the positions of the 18cysteine residues in the gp120 primary sequence, and the positions of 13of the approximately 23 N-linked glycosylation sites in the gp120sequence.

The ordinarily skilled worker may use the disulfide bonding patternwithin gp120 and the positions of actual oligosaccharide moieties on themolecule for directing mutagenesis and fragmentation variants. It isintended that the variants of this invention include unclipped HIV envin which one or more residues--other than those at the clip site--havebeen substituted in the env amino acid sequence, deletions of one ormore residues in the env sequence other than the clip site, andinsertions of one or more residues adjacent to any residues exceptwithin the clip site.

Lasky et al., Science 233:209-212 (1986) described the expression ofgp120 in Chinese hamster ovary (CHO) cells as a fusion protein using thesignal peptide of the herpes simplex type 1 glycoprotein D (gD1). Theuse of two such fusions proteins are preferred in the practice of thisinvention. A recombinant glycoprotein (CL44) is expressed as a 498-aminoacid fusion protein containing the first 27 residues of gD1 fused toresidues 31-501 of gp120. This construction lacks the first cysteineresidue of mature gp120. Another preferred recombinant fusion protein(9AA) contains the first 9 residues of gD1 fused to residues 4-501 ofgp120. This restores the first cysteine residue, Cys24. Carboxy-terminalanalysis of CL44 using carboxypeptidase digestions indicate thatglutamic acid residue 479 is the carboxy terminus of the fully processedmolecule secreted by CHO cells (data not shown). The amino acidsequences of these two constructions are given in FIG. 2.

This invention also contemplates amino acid sequence variants of theunclipped HIV env. Amino acid sequence variants are prepared withvarious objectives in mind, including increasing the affinity of theunclipped HIV env for a ligand or antibody, facilitating the stability,purification and preparation of the unclipped HIV env, modifying itsplasma half life, improving therapeutic efficacy, and lessening theseverity or occurrence of side effects during therapeutic use of theunclipped HIV env. In the discussion below, amino acid sequence variantsof the unclipped HIV env are provided, exemplary of the variants thatmay be selected.

Amino acid sequence variants of unclipped HIV env fall into one or moreof three classes: Insertional, substitutional, or deletional variants.These variants ordinarily are prepared by site-specific mutagenesis ofnucleotides in the DNA encoding the unclipped HIV env, by which DNAencoding the variant is obtained, and thereafter expressing the DNA inrecombinant cell culture. However, fragments having up to about 100-150amino acid residues are prepared conveniently by in vitro synthesis. Thefollowing discussion applies to any unclipped HIV env to the extent itis applicable to its structure or function.

The amino acid sequence variants of the unclipped HIV env arepredetermined variants not found in nature or naturally occurringalleles. The unclipped HIV env variants typically exhibit the samequalitative biological--for example, antibody binding--activity as thenaturally occurring unclipped HIV env or unclipped HIV env analogue.However, unclipped HIV env variants and derivatives that are not capableof binding to antibodies are useful nonetheless (a) as a reagent indiagnostic assays for HIV env or antibodies to the HIV env, (b) wheninsolubilized in accord with known methods, as agents for purifyinganti-unclipped HIV env antibodies from antisera or hybridoran culturesupernatants, and (c) as immunogens for raising antibodies to unclippedHIV env or as immunoassay kit components (labelled, as a competitivereagent for the native HIV env or unlabelled as a standard for HIV envassay) so long as at least one HIV env epitope remains active.

While the site for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random or saturation mutagenesis (where all 20 possible residuesare inserted) is conducted at the target codon and the expressed HIV envvariant is screened for the optimal combination of desired activities.Such screening is within the ordinary skill in the art.

Amino acid insertions usually will be on the order of about from 1 to 10amino acid residues; substitutions are typically introduced for singleresidues; and deletions will range about from 1 to 30 residues.Deletions or insertions preferably are made in adjacent pairs, i.e. adeletion of 2 residues or insertion of 2 residues. It will be amplyapparent from the following discussion that substitutions, deletions,insertions or any combination thereof are introduced or combined toarrive at a final construct.

Insertional amino acid sequence variants of unclipped HIV env are thosein which one or more amino acid residues extraneous to the HIV env areintroduced into a predetermined site (other than at the clip site) inthe target unclipped HIV env and which displace the preexistingresidues.

Commonly, insertional variants are fusions of heterologous proteins orpolypeptides to the amino or carboxyl terminus of the unclipped HIV env.Such variants are referred to as fusions of the unclipped HIV env and apolypeptide containing a sequence which is other than that which isnormally found in the unclipped HIV env at the inserted position.Several groups of fusions are contemplated herein.

The novel polypeptides of this invention are useful in diagnostics or inpurification of the antibodies or ligands by known immunoaffinitytechniques.

Desirable fusions of unclipped HIV env, which may or may not also beimmunologically active, include fusions of the mature unclipped HIV envsequence with a signal sequence heterologous to the binding partner asmentioned above. Signal sequence fusions are employed in order to moreexpeditiously direct the secretion of the unclipped HIV env. Theheterologous signal replaces the native HIV env signal, and when theresulting fusion is recognized, i.e. processed and cleaved by the hostcell, the unclipped HIV env is secreted. Signals are selected based onthe intended host cell, and may include bacterial yeast, mammalian andviral sequences. The native HIV env signal or the herpes gD glycoproteinsignal is suitable for use in mammalian expression systems.

C-terminal or N-terminal fusions of the unclipped HIV env or unclippedHIV env fragment with an immunogenic hapten or heterologous polypeptideare useful as vaccine components for the immunization of patientsagainst HIV infection. Fusions of the hapten or heterologous polypeptidewith unclipped HIV env or its active fragments which retain T-cellbinding activity are also useful in directing cytotoxic T cells againsttarget cells where the hapten or heterologous polypeptide is capable ofbinding to a target cell surface receptor. For example, membrane-boundtransforming growth factor-α(TGF-α) is present on the surface of manysolid (non-hematopoietic) neoplastic tumors. Antibodies capable ofbinding TGF-α are known, and may be linked to unclipped HIV env, e.g. bycovalent crosslinking according to commonly known methods, or byexpression in recombinant cell culture as an N- or C-terminal fusionwith unclipped HIV env or active fragment, and are used to targetunclipped gp120 to TGF-α.

The precise site at which the fusion is made is variable; particular HIVenv sites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of theunclipped HIV env. The optimal site will for a particular applicationwill be determined by routine experimentation.

Substitutional variants are those in which at least one residue in theFIG. 1 or 2 sequence has been removed (other than those residues at theclip site) and a different residue inserted in its place. Suchsubstitutions generally are made in accordance with the following Table1 when it is desired to finely modulate the characteristics of theunclipped HIV env.

                  TABLE 1                                                         ______________________________________                                        Original Residue  Exemplary Substitutions                                     ______________________________________                                        Ala               ser                                                         Arg               lys                                                         Asn               gln; his                                                    Asp               glu                                                         Cys               ser; ala                                                    Gln               asn                                                         Glu               asp                                                         Gly               pro                                                         His               asn; gln                                                    Ile               leu; val                                                    Leu               ile; val                                                    Lys               arg; gln; glu                                               Met               leu; ile                                                    Phe               met; leu; tyr                                               Ser               thr                                                         Thr               ser                                                         Trp               tyr                                                         Tyr               trp; phe                                                    Val               ile; leu                                                    ______________________________________                                    

Novel amino acid sequences, as well as isosteric analogs (amino acid orotherwise), as included within the scope of this invention.

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in unclipped HIVenv properties will be those in which (a) a hydrophilic residue, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine.

Some deletions, insertions, and substitutions will not produce radicalchanges in the characteristics of the unclipped HIV env molecule.However, when it is difficult to predict the exact effect of thesubstitution, deletion, or insertion in advance of doing so, for examplewhen modifying an immune epitope, one skilled in the art will appreciatethat the effect will be evaluated by routine screening assays. Forexample, a variant typically is made by site specific mutagenesis of theHIV env -encoding nucleic acid, expression of the variant nucleic acidin recombinant cell culture and, optionally, purification from the cellculture for example by immunoaffinity adsorption on a polyclonalanti-unclipped HIV env column (in order to adsorb the variant by atleast one remaining immune epitope). The activity of the cell lysate orpurified unclipped HIV env variant is then screened in a suitablescreening assay for the desired characteristic. For example, a change inthe immunological character of the unclipped HIV env, such as affinityfor T-cell binding, measured by a competitive-type immunoassay. As morebecomes known about the functions in vivo of the unclipped HIV env otherassays will become useful in such screening. Modifications of suchprotein properties as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, or the tendency to aggregatewith carriers or into multimers are assayed by methods well known to theartisan.

Another class of unclipped HIV env variants are deletional variants.Deletions are characterized by the removal of one or more amino acidresidues (other than the clip site) from the HIV env sequence.Typically, deletions are used to affect unclipped HIV env biologicalactivities, however, deletions which preserve the biological activity orimmune cross-reactivity of the unclipped HIV env are suitable.

Deletions of cysteine or other labile residues also may be desirable,for example in increasing the oxidative stability of the unclipped HIVenv. Deletion or substitutions of potential proteolysis sites, e.g. ArgArg, is accomplished by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

It will be understood that some variants may exhibit reduced or absentbiological activity. These variants nonetheless are useful as standardsin immunoassays for HIV env so long as they retain at least one immuneepitope of HIV env.

It is presently believed that the three-dimensional structure of theunclipped HIV env compositions of the present invention is important totheir functioning as described herein. Therefore, all related structuralanalogs which mimic the active structure of those formed by thecompositions claimed herein are specifically included within the scopeof the present invention.

Glycosylation variants are included within the scope of unclipped HIVenv. They include variants completely lacking in glycosylation(unglycosylated) and variants having at least one less glycosylated sitethan the native form (deglycosylated) as well as variants in which theglycosylation has been changed. Included are deglycosylated andunglycosylated amino acid sequence variants, deglycosylated andunglycosylated unclipped HIV env having the native, unmodified aminoacid sequence of HIV env, and other glycosylation variants. For example,substitutional or deletional mutagenesis is employed to eliminate the N-or O-linked glycosylation sites of unclipped HIV env, e.g., anasparagine residue (not at the clip site) is deleted or substituted forby another basic residue such as lysine or histidine. Alternatively,flanking residues making up the glycosylation site are substituted ordeleted, even though the asparagine residues remain unchanged, in orderto prevent glycosylation by eliminating the glycosylation recognitionsite.

Unglycosylated unclipped HIV env which has the amino acid sequence ofthe native HIV env is produced in recombinant prokaryotic cell culturebecause prokaryotes are incapable of introducing glycosylation intopolypeptides.

Glycosylation variants are produced by selecting appropriate host cellsor by in vitro methods. Yeast, for example, introduce glycosylationwhich varies significantly from that of mammalian systems. Similarly,mammalian cells having a different species (e.g. hamster, murine,insect, porcine, bovine or ovine) or tissue origin (e.g. lung, liver,lymphold, mesenchymal or epidermal) than the source of the HIV envantigen are routinely screened for the ability to introduce variantglycosylation as characterized for example by elevated levels of mannoseor variant ratios of mannose, fucose, sialic acid, and other sugarstypically found in mammalian glycoproteins. In vitro processing of theunclipped HIV env typically is accomplished by enzymatic hydrolysis,e.g. neuraminidase digestion.

Covalent modifications of the unclipped HIV env molecule which do notmodify the clip site are included within the scope hereof. Suchmodifications are introduced by reacting targeted amino acid residues ofthe recovered protein with an organic derivatizing agent that is capableof reacting with selected side chains or terminal residues, or byharnessing mechanisms of post-translational modification that functionin selected recombinant host cells. The resulting covalent derivativesare useful in programs directed at identifying residues important forbiological activity, for immunoassays of HIV env or for the preparationof anti-unclipped HIV env antibodies for immunoaffinity purification ofthe recombinant unclipped HIV env. For example, complete inactivation ofthe biological activity of the protein after reaction with ninhydrinwould suggest that at least one arginyl or lysyl residue is critical forits activity, whereafter the individual residues which were modifiedunder the conditions selected are identified by isolation of a peptidefragment containing the modified amino acid residue. Such modificationsare within the ordinary skill in the art and are performed without undueexperimentation.

Derivatization with bifunctional agents is useful for preparingintermolecular aggregates of the unclipped HIV env with polypeptides aswell as for cross-linking the unclipped HIV env to a water insolublesupport matrix or surface for use in the assay or affinity purificationof its ligands. In addition, a study of intrachain cross-links willprovide direct information on conformational structure. Commonly usedcross-linking agents include suflhydryl reagents,1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde,N-hydroxysuccinimideesters,forexample esters with 4-azidosalicylic acid, homobifunctional imidoestersincluding disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl-propionate), and bifunctional maleimides such asbis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-(p-azido-phenyl)dithio! propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,080; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Polymers generally are covalently linked to the peptide herein through amultifunctional crosslinking agent which reacts with the polymer and oneor more amino acid or sugar residues of protein. However, it is withinthe scope of this invention to directly crosslink the polymer byreacting a derivatized polymer with the peptide, or vice versa. Covalentbonding to amino groups is accomplished by known chemistries based uponcyanuric chloride, carbonyl diimidazole, aldehyde reactive groups (PEGalkoxide plus diethyl acetal of bromoacetaldehyde; PEG plus DMSO andacetic anhydride, or PEG chloride plus the phenoxide of4-hydroxybenzaldehyde, succinimidyl active esters, activateddithiocarbonate PEG, 2,4,5-trichlorophenylchloroformate orp-nitrophenylchloroformate activated PEG. Carboxyl groups arederivatized by coupling PEG-amine using carbodiimide.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-861983!), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

DNA encoding unclipped HIV env is synthesized by in vitro methods or isobtained readily from cDNA libraries. The means for synthetic creationof the DNA encoding unclipped HIV env, either by hand or with anautomated apparatus, are generally known to one of ordinary skill in theart, particularly in light of the teachings contained herein. Asexamples of the current state of the art relating to polynucleotidesynthesis, one is directed to Maniatis et al., Molecular Cloning--ALaboratory Manual, Cold Spring Harbor Laboratory (1984), and Horvath etal., An Automated DNA Synthesizer Employing Deoxynucleoside3'-Phosphoramidites, Methods in Enzymology 154:313-326, 1987, herebyspecifically incorporated by reference.

Alternatively, to obtain DNA encoding unclipped HIV env, one needs onlyto conduct hybridization screening with labelled DNA encoding either theHIV env or unclipped HIV env fragments thereof as shown in FIGS. 1 and 2(usually, greater than about 20, and ordinarily about 50 bp) in order todetect clones which contain homologous sequences in the cDNA librariesderived from cells or tissues of a particular animal, followed byanalyzing the clones by restriction enzyme analysis and nucleic acidsequencing to identify full-length clones. If full length clones are notpresent in the library, then appropriate fragments are recovered fromthe various clones and ligated at restriction sites common to thefragments to assemble a full-length clone. DNA encoding HIV env fromvarious isotypes and strains is obtained by probing libraries from hostsof such species with the sequences of FIGS. 1 or 2, or by synthesizingthe genes in vitro. DNA for other isotypes or strains having knownsequences may be obtained with the use of analogous routinehybridization procedures.

In general, prokaryotes are used for cloning of DNA sequences inconstructing the vectors useful in the invention. For example, E. coliK12 strain 294 (ATCC No. 31446) is particularly useful. Other microbialstrains which may be used include E. coli B and E. coli X1776 (ATCC No.31537). These examples are illustrative rather than limiting.Alternatively, in vitro methods of cloning, e.g. polymerase chainreaction, are suitable.

The polypeptides of this invention are expressed directly in recombinantcell culture as an N-terminal methionyl analogue, or as a fusion with apolypeptide heterologous to the hybrid/portion, preferably a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the hybrid/portion. For example, in constructing aprokaryotic secretory expression vector for unclipped HIV env, thenative HIV env signal is employed with hosts that recognize that signal.When the secretory leader is "recognized" by the host, the host signalpeptidase is capable of cleaving a fusion of the leader polypeptidefused at its C-terminus to the desired mature unclipped HIV env. Forhost prokaryotes that do not process the HIV env signal, the signal issubstituted by a prokaryotic signal selected for example from the groupof the alkaline phosphatase, penicillinase, 1 pp or heat stableenterotoxin II leaders. For yeast secretion the HIV env signal may besubstituted by the yeast invertase, alpha factor or acid phosphataseleaders. In mammalian cell expression the native signal is satisfactoryfor unclipped mammalian HIV env, although other mammalian secretoryprotein signals are suitable, as are viral secretory leaders, forexample the herpes simplex gD signal.

Unclipped HIV env may be expressed in any host cell, but preferably issynthesized in mammalian hosts. However, host cells from prokaryotes,fungi, yeast, insects and the like are also are used for expression.Exemplary prokaryotes are the strains suitable for cloning as well as E.coli W3110 (F⁻ 'λ⁻ ' prototrophic, ATTC No. 27325), otherenterobacteriaceae such as Serratia marcescans, bacilli and variouspseudomonads. Preferably the host cell should secrete minimal amounts ofproteolytic enzymes.

Expression hosts typically are transformed with DNA encoding theunclipped HIV env which has been ligated into an expression vector. Suchvectors ordinarily carry a replication site (although this is notnecessary where chromosomal integration will occur). Expression vectorsalso include marker sequences which are capable of providing phenotypicselection in transformed cells, as will be discussed further below. Forexample, E. coli is typically transformed using pBR322, a plasmidderived from an E. coli species (Bolivar, et al., Gene 2:95 1977!).pBR322 contains genes for ampicillin and tetracycline resistance andthus provides easy means for identifying transformed cells, whether forpurposes of cloning or expression. Expression vectors also optimallywill contain sequences which are useful for the control of transcriptionand translation, e.g., promoters and Shine-Dalgarno sequences (forprokaryotes) or promoters and enhancers (for mammalian cells). Thepromoters may be, but need not be, inducible; even powerful constitutivepromoters such as the CMV promoter for mammalian hosts may produceunclipped HIV env without host cell toxicity. While it is conceivablethat expression vectors need not contain any expression control,replicative sequences or selection genes, their absence may hamper theidentification of transformants and the achievement of high levelpeptide expression.

Promoters suitable for use with prokaryotic hosts illustratively includethe β-lactamase and lactose promoter systems (Chang et al., Nature275:615 1978!; and Goeddel et al., Nature 281:544 1979!), alkalinephosphatase, the tryptophan (trp) promoter system (Goeddel, NucleicAcids Res. 8:4057 (1980) and EPO Appln. Publ. No. 35,776) and hybridpromoters such as the tac promoter (H. de Boer et al., Proc. Natl. Acad.Sci. USA 80:21-25 1983!). However, other functional bacterial promotersare suitable. Their nucleotide sequences are generally known, therebyenabling a skilled worker operably to ligate them to DNA encodingunclipped HIV env. (Siebenlist et al., Cell 20:269 1980!) using linkersor adaptors to supply any required restriction sites. Promoters for usein bacterial systems also will contain a Shine-Dalgarno (S. D.) sequenceoperably linked to the DNA encoding the unclipped HIV env.

In addition to prokaryotes, eukaryotic microbes such as yeast orfilamentous fungi are satisfactory. Saccharomyces cerevisiae is the mostcommonly used eukaryotic microorganism, although a number of otherstrains are commonly available. The plasmid YRp7 is a satisfactoryexpression vector in yeast (Stinehcomb, et al., Nature 282:39 (1979);Kingsman et al, Gene 7:141 (1979); Tschemper et al., Gene 10:157(1980)). This plasmid already contains the trp1 gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones,Genetics 85:12 1977!). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1968); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., European Patent Publication No. 73,657A.

Expression control sequences are known for eucaryotes. Virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence which may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are inserted into mammalian expressionvectors.

Suitable promoters for controlling transcription from vectors inmammalian host cells are readily obtained from various sources, forexample, the genomes of viruses such as polyoma virus, SV40, adenovirus,MMV (steroid inducible), retroviruses (e.g. the LTR of HIV), hepatitis-Bvirus and most preferably cytomegalovirus, or from heterologousmammalian promoters, e.g. the beta actin promoter. The early and latepromoters of SV40 are conveniently obtained as an SV40 restrictionfragment which also contains the SV40 viral origin of replication. Fierset al., Nature, 273:113 (1978). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment. Greenaway, P. J. et al., Gene 18:355-360 (1982).

Transcription of a DNA encoding unclipped HIV env by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10-300 bp,that act on a promoter to increase its transcription. Enhancers arerelatively orientation and position independent having been found 5'(Laimins et al., PNAS 78:993 1981!) and 3' (Lusky, M. L., et al., Mol.Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron(Banerji, J. L. et al., Cell 33:729 (1983)) as well as within the codingsequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4:1293 1984!).Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (bp 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription which may affect mRNA expression. These regions aretranscribed as polyadenylated segments in the untranslated portion ofthe mRNA encoding the hybrid immunoglobulin. The 3' untranslated regionsalso include transcription termination sites.

Expression vectors may contain a selection gene, also termed aselectable marker. Examples of suitable selectable markers for mammaliancells are dihydrofolate reductase (DHFR), thymidine kinage (TK) orneomycin. When such selectable markers are successfully transferred intoa mammalian host cell, the transformed mammalian host cell is able tosurvive if placed under selective pressure. There are two widely useddistinct categories of selective regimes. The first category is based ona cell's metabolism and the use of a mutant cell line which lacks theability to grow independent of a supplemented media. Two examples areCHO DHFR⁻ cells and mouse LTK⁻ cells. These cells lack the ability togrow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media. Inpreferred embodiments, herein, CHO cells which are DHFR⁺ are used forrecombinant expression of gp120.

The second category of selective regimes is dominant selection whichrefers to a selection scheme used in any cell type and does not requirethe use of a mutant cell line. These schemes typically use a drug toarrest growth of a host cell. Those cells which are successfullytransformed with a heterologous gene express a protein conferring drugresistance and thus survive the selection regimen. Examples of suchdominant selection use the drugs neomycin (Southern et al., J. Molec.Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan et al., Science209:1422 (1980)) or hygromycin (Sugden et al., Mol. Cell. Biol.5:410-413 (1985)). The three examples given above employ bacterial genesunder eukaryotic control to convey resistance to the appropriate drugG418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin,respectively.

"Amplification" refers to the increase or replication of an isolatedregion within a cell's chromosomal DNA. Amplification is achieved usinga selection agent, e.g. methotrexate (MTX) which inactivates DHFR.Amplification or the making of successive copies of the DHFR generesults in greater amounts of DHFR being produced in the face of greateramounts of MTX. Amplification pressure is applied notwithstanding thepresence of endogenous DHFR, by adding ever greater amounts of MTX tothe media. Amplification of a desired gene can be achieved bycotransfecting a mammalian host cell with a plasmid having a DNAencoding a desired protein and teh DHFR or amplification gene permittingcointegration. One ensures that the cell requires more DHFR, whichrequirement is met by replication of teh selection eerie, by selectingonly for cells that can grow in teh presence of ever-greater MTXconcentration. So long as the gene encoding a desired heterologousprotein has cointegrated with the selection gene replication of thisgene gives rise to replication of the gene encoding the desired protein.The result is that increased copies of the gene, i.e. an amplified gene,encoding the desired heterologous protein express more of the desiredprotein.

Suitable eukaryotic host cells for expressing unclipped HIV env includemonkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham, F. L. et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamsterovary-cells-DHFR (CHO, Urlaub and Chasin, PNAS (USA) 77:4216, 1980!);mouse sertoli cells (TM4, Mather, J. P., Biol. Reprod. 23:243-2511980!); monkey kidney cells (CV1 ATCC CCL 70); african green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); and, TRI cells (Mather, J. P. et al., Annals N.Y.Acad. Sci. 383:44-68 1982!).

Construction of suitable vectors containing the desired coding andcontrol sequences employ standard ligation techniques. Isolated plasmidsor DNA fragments are cleaved, tailored, and religated in the formdesired to form the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction and/or sequenced bythe method of Messing et al., Nucleic Acids Res. 9:309 (1981 ) or by themethod of Maxam et al., Methods in Enzymology 65:499 (1980).

Host cells are transformed with the expression vectors of this inventionand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants or amplifying the genesencoding the desired sequences. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

To maximize the harvest of unclipped HIV env, it is currently preferredthat cell cultures be grown in media containing low serum, preferably0-3 percent serum, and more preferably in about 1 percent fetal bovineserum or other suitable serum.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells which are within a host animal.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or by chromosomalintegration. Unless indicated otherwise, the method used herein fortransformation of the host cells is the method of Graham, F. and van derEb, A., Virology 52:456-457 (1973). However, other methods forintroducing DNA into cells such as by nuclear injection or by protoplastfusion may also be used. If prokaryotic cells or cells which containsubstantial cell wall constructions are used, the preferred method oftransfection is calcium treatment using calcium chloride as described byCohen, F. N. et al., Proc. Natl. Acad. Sci. (USA), 69:2110 (1972).

"Transfection" refers to the introduction of DNA into a host cellwhether or not any coding sequences are ultimately expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Transformation of the host cell isthe indicia of successful transfection.

The novel polypeptide of this invention is recovered and purified fromrecombinant cell cultures by known methods, including ammonium gulf ateor ethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, immunoaffinitychromatography, hydroxyapatite chromatography and lectin chromatography.See, e.g., the purification methods described in EP 187,041. Moreover,reverse-phase HPLC and chromatography using ligands for unclipped HIVenv are useful for purification. It is presently preferred to utilizegel permeation chromatography and anion exchange chromatography, andmore preferred to use cation exchange and hydrophobic interactionchromatography (HIC) according to standard protocols.

Additionally, unclipped HIV env is recovered and purified by passageover a column of unclipped HIV env-antibody covalently coupled toaldehyde silica by a standard procedure (Roy et al., Journal ofChromatography 303:225-228(1984)), washing of the column with a salinesolution, and analyzing the eluant by standard methods such asquantitative amino acid analysis. Procedures utilizing monoclonalantibodies coupled to glycerol-coated controlled pore glass aredesirable for the practice of this invention. Optionally, lowconcentrations (approximately 1-5 mM) of calcium ion may be presentduring purification. Unclipped HIV env may preferably be purified in thepresence of a protease inhibitor such as PMSF.

Unclipped HIV env is placed into pharmaceutically acceptable, sterile,isotonic formulations together with required cofactors, and optionallyare administered by standard means well known in the field. Theformulation is preferably liquid, and is ordinarily a physiologic saltsolution containing non-phosphate buffer at pH 6.8-7.6, or may belyophilized powder.

The unclipped HIV env compositions to be used in the therapy will beformulated and dosages established in a fashion consistent with goodmedical practice taking into account the disorder to be treated, thecondition of the individual patient, the site of delivery of theunclipped HIV env polypeptide, the method of administration and otherfactors known to practitioners.

Unclipped HIV env is prepared for administration by mixing unclipped HIVenv at the desired degree of purity with adjuvants or physiologicallyacceptable carriers i.e. carriers which are nontoxic to recipients atthe dosages and concentrations employed. Adjuvants and carriers aresubstances that in themselves share no immune epitopes with the targetantigen, but which stimulate the immune response to the target antigen.Ordinarily, this will entail combining unclipped HIV env with buffers,low molecular weight (less that about 10 residues) polypeptides,proteins, amino acids, carbohydrates including glucose or dextrans,chelating agents such as EDTA, and other excipients. Freunds adjuvant (amineral oil emulsion) commonly has been used for this purpose, as have avariety of toxic microbial substances such as mycobacterial extracts andcytokines such as tumor necrosis factor and interferon gamma (describedin co-pending U.S. Ser. No. 07/007,075). Although antigen is desirablyadministered with an adjuvant, in sitatuions where the initialinoculation is delivered with an adjuvant, boosters with antigen may notrequire adjuvant. Carriers often act as adjuvants, but are generallydistinguished from adjuvants in that carriers comprise water insolublemacromolecular particulate structures which aggregate the antigen,Typical carriers include aluminum hydroxide, latex particles, bentoniteand liposomes.

It is envisioned that injections (intramuscular or subcutaneous) will bethe primary route for therapeutic administration of the vaccines of thisinvention, intravenous delivery, or delivery through catheter or othersurgical tubing is also used. Alternative routes include tablets and thelike, commercially available nebulizers for liquid formulations, andinhalation of lyophilized or aerosolized receptors. Liquid formulationsmay be utilized after reconstitution from powder formulations.

The novel polypeptide may also be administered via microspheres,liposomes, other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semipermeable polymermatrices in the form of shaped articles, e.g. suppositories, ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919, EP 58,481 ) copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al.,Biopolymers 22(1):547-556, (1985)), poly (2-hydroxyethyl-methacrylate)or ethylene vinyl acetate (R. Langer et al., J. Biomed. Mater. Res.15:167-277 (1981) and R. Langer, Chem. Tech. 12:98-105 (1982)).Liposomes containing the unclipped HIV env are prepared by well-knownmethods: DE 3,218,121 A; Epstein et al., Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,77:4030-4034 (1980); EP 52322A; EP 36676A; EP 88046A; EP 143949A; EP142541A; Japanese patent application 83-11808; U.S. Pat. Nos. 4,485,045and 4,544,545; and UP 102,342A. Ordinarily the liposomes are of thesmall (about 200-800 Angstroms) unilamelar type in which the lipidcontent is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal rate of the polypeptideleakage.

The vaccination dose of the unclipped HIV env administered will bedependent upon the properties of the vaccine employed, e.g. its bindingactivity and in vivo plasma half-life, the concentration of theunclipped HIV env in the formulation, the administration route, the siteand rate of dosage, the clinical tolerance of the patient involved, thepathological condition afflicting the patient and the like, as is wellwithin the skill of the physician. Generally, doses of 300 μg ofunclipped HIV env per patient per administration are preferred, althoughdosages may range from about 10 μg-1 mg per dose. Different dosages areutilized during a series of sequential inoculations; the practitionermay administer an initial inoculation and then boost with relativelysmaller doses of unclipped HIV env vaccine.

The unclipped HIV env vaccines of this invention may be administered ina variety of ways and to different classes of recipients. The vaccinesare used to vaccinate individuals who may or may not be at risk ofexposure to HIV, and additionally, the vaccines are desirablyadministered to seropositive individuals and to individuals who havebeen previously exposed to HIV (see. e.g. Salk, Nature 327:473-476(1987); and Salk et al., Science 195:834-847 (1977)).

The unclipped HIV env may be administered in combination with otherantigens in a single inoculation "cocktail". The unclipped HIV envvaccines may also be administered as one of a series of inoculationsadministered over time. Such a series may include inoculation with thesame or different preparations of HIV antigens or other vaccines.

The adequacy of the vaccination parameters chosen, e.g. dose, schedule,adjuvant choice and the like, is determined by taking aliquots of serumfrom the patient and assaying antibody titers during the course of theimmunization program. Alternatively, the presence of T cells may bymonitored by conventional methods as described in Example 1 below. Inaddition, the clinical condition of the patient will be monitored forthe desired effect, e.g. anti-infective effect. If inadequatevaccination is achieved then the patient can be boosted with furtherunclipped HIV env vaccinations and the vaccination parameters can bemodified in a fashion expected to potentiate the immune response, e.g.increase the amount of antigen and/or adjuvant, complex the antigen witha carrier or conjugate it to an immunogenic protein, or vary the routeof administration.

This invention is also directed to optimized immunization schedules forenhancing a protective immune response against HIV infection. It iscurrently preferred that at least three separate inoculations withunclipped HIV env be administered, with a second inoculation beingadministered more than two, preferably three to eight, and morepreferably approximately four weeks following the first inoculation. Itis preferred that a third inoculation be administered several monthslater than the second "boost" inoculation, preferably at least more thanfive months following the first inoculation, more preferably six monthsto two years following the first inoculation, and even more preferablyeight months to one year following the first inoculation. Periodicinoculations beyond the third are also desirable to enhance thepatient's "immune memory". See Anderson et al., J. Infectious Diseases160(6):960-969 (December 1989) and the references therein, incorporatedby reference herein. Generally, infrequent immunizations with unclippedHIV env spaced at relatively long intervals is more preferred thanfrequent immunizations in eliciting maximum antibody responses, and ineliciting a protective effect.

The polypeptides of this invention may optionally be administered alongwith other pharmacologic agents used to treat AIDS or ARC or otherHIV-related diseases and infections, such as AZT, CD4, antibiotics,immunomodulators such as interferon, anti-inflammatory agents, andanti-tumor agents.

Antibodies

This invention is also directed to monoclonal antibodies, as illustratedin Example 2 below and by antibody hybridomas deposited with the ATCC(as described below). In accordance with this invention, monoclonalantibodies specifically binding an epitope of unclipped HIV env orantigenically active, cell surface-exposed fragments thereof (forexample epitopes chosen from gp120, gp41 ) are isolated from continuoushybrid cell lines formed by the fusion of antigen-primed immunelymphocytes with myeloma cells. Advantageously, the monoclonalantibodies of the subject invention which bind HIV env and unclipped HIVenv bind the domain of this protein which is exposed on the cellsurface.

The antibodies of the subject invention are obtained through routinescreening. An assay is used for screening monoclonal antibodies fortheir cytotoxic potential as ricin A chain containing immunotoxins. Theassay involves treating cells with dilutions of the test antibodyfollowed by a Fab fragment of a secondary antibody coupled to ricin Achain (`indirect assay`). The cytotoxicity of the indirect assay iscompared to that of the direct assay where the monoclonal antibody iscoupled to ricin A chain. The indirect assay accurately predicts thepotency of a given monoclonal antibody as an immunotoxin and is thususeful in screening monoclonal antibodies for use as immunotoxins--seealso Vitetta et al, Science 238:1098-1104 (1987), and Weltman et al.,Cancer Res. 47:5552 (1987), hereby incorporated by reference.

Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional antibody(polyclonal) preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.Monoclonal antibodies are useful to improve the selectivity andspecificity of diagnostic and analytical assay methods usingantigen-antibody binding. A second advantage of monoclonal antibodies isthat they are synthesized by the hybridoma culture, uncontaminated byother immunoglobulins. Monoclonal antibodies may be prepared fromsupernatants of cultured hybridoma cells or from ascites induced byintra-peritoneal inoculation of hybridoma cells into mice.

The hybridoma technique described originally by Kohler and Milstein,Eur. J. Immunol., 6:511 (1976) has been widely applied to produce hybridcell lines that secrete high levels of monoclonal antibodies againstmany specific antigens.

In particular embodiments of this invention, an antibody capable ofblocking or binding to an epitope spanning the gp120 proteolytic clipsite is obtained by immunizing mice such as Balb/c or, preferably C57BL/6, against gp120 and screening for a clonal antibody that, whenpreincubated with unclipped gp120, prevents its binding to clippedgp120. The 10F6, 11G5, and 10D8 antibodies are examples of such anantibody, but they are not unique since other antibodies having the samequalitative activity can be obtained by the method described herein, andas described above, antibodies which span the clip site are known in theliterature. "Qualitative" activity in this context means that theantibody binds to an epitope spanning the clip site of HIV env. Atryptic digest of gp120 showed that the 10F6, 11G5, and 10D8 antibodiesbound the peptide spanning residues approximately 301-324, and thus spanthe clip site. Monoclonal antibodies other than 10F6, 11G5, and 10D8which are capable of doing so are useful in the practice of thisinvention, despite any differences in affinity, immunoglobulin class,species of origin, or gp120 epitope, as are antibodies which areexpressed in recombinant cell culture or that are predetermined aminoacid sequence variants of the 10F6, 11G5, and 10D8 antibodies, e.g.chimeras of the variable region of the 10F6, 11G5, and 10D8 antibodiesand a human constant region.

The route and schedule of immunization of the host animal or culturedantibody-producing cells therefrom are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction. Applicants have employed mice as the test model although itis contemplated that any mammalian subject including human subjects orantibody producing cells therefrom can be manipulated according to theprocesses of this invention to serve as the basis for production ofmammalian, including human, hybrid cell lines.

After immunization, immune lymphold cells are fused with myeloma cellsto generate a hybrid cell line which can be cultivated and subcultivatedindefinitely, to produce large quantities of monoclonal antibodies. Forpurposes of this invention, the immune lymphold cells selected forfusion are lymphocytes and their normal differentiated progeny, takeneither from lymph node tissue or spleen tissue from immunized animals.Applicants prefer to employ immune spleen cells, since they offer a moreconcentrated and convenient source of antibody producing cells withrespect to the mouse system. The myeloma cells provide the basis forcontinuous propagation of the fused hybrid. Myeloma cells are tumorcells derived from plasma cells.

It is possible to fuse cells of one species with another. However, it ispreferred that the source of immunized antibody producing cells andmyeloma be from the same species.

The hybrid cell lines can be maintained in culture in vitro in cellculture media. The cell lines of this invention can be selected and/ormaintained in a composition comprising the continuous cell line inhypoxanthine-aminopterin thymidine (HAT) medium. In fact, once thehybridoma cell line is established, it can be maintained on a variety ofnutritionally adequate media. Moreover, the hybrid cell lines can bestored and preserved in any number of conventional ways, includingfreezing and storage under liquid nitrogen. Frozen cell lines can berevived and cultured indefinitely with resumed synthesis and secretionof monoclonal antibody. The secreted antibody is recovered from tissueculture supernatant by conventional methods such as precipitation, Ionexchange chromatography, affinity chromatography, or the like. Theantibodies described herein are also recovered from hybridoma cellcultures by conventional methods for purification of IgG or IgM as thecase may be that heretofore have been used to purify theseimmunoglobulins from pooled plasma, e.g. ethanol or polyethylene glycolprecipitation procedures. The purified antibodies are sterile filtered,and optionally are conjugated to a detectable marker such as an enzymeor spin label for use in diagnostic assays of HIV in test samples.

While the invention is demonstrated using mouse monoclonal antibodies,the invention is not so limited; in fact, human antibodies may be usedand may prove to be preferable. Such antibodies can be obtained by usinghuman hybridomas (Cote et al., Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, p. 77 (1985)). In fact, according to the invention,techniques developed for the production of chimeric antibodies (Morrisonet al., Proc. Natl. Acad. Sci., 81:6851 (1984); Neuberger et al., Nature312:604 (1984); Takeda et al., Nature 314:452 (1985)) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity (such as ability to activate human complement andmediate ADCC) can be used; such antibodies are within the scope of thisinvention.

As another alternative to the cell fusion technique, EBV-immortalized Bcells are used to produce the monoclonal antibodies of the subjectInvention. Other methods for producing monoclonal antibodies such asrecombinant DNA, are also contemplated.

Immunotoxins

This invention is also directed to immunochemical derivatives of theantibodies of this invention such as immunotoxins (conjugates of theantibody and a cytotoxic moiety). The antibodies are also used to inducelysis through the natural complement process, and to interact withantibody dependent cytotoxic cells normally present.

Purified, sterile filtered antibodies are optionally conjugated to acytotoxin such as ricin for use in AIDS therapy. U.S. patent applicationSer. No. 07/350,895 illustrates methods for making and usingimmunotoxins for the treatment of HIV infection, and its teachings arespecifically incorporated by reference herein.

Immunotoxins of this invention, capable of specifically binding HIV env,are used to kill cells that are already infected and are activelyproducing new virus. Killing is accomplished by the binding of theimmunotoxin to viral coat protein which is expressed on infected cells.The immunotoxin is then internalized and kills the cell. Infected cellsthat have incorporated viral genome into their DNA but are notsynthesizing vital protein (i.e., cells in which the virus is latent)may not be susceptible to killing by immunotoxin until they begin tosynthesize virus. The antibodies of this invention which span the clipsite and/or the other antibodies described herein may be used alone orin any combination with for delivering toxins to infected cells. Inaddition, a toxin-antibody conjugate can bind to circulating viruses orviral coat protein which will then effect killing of cells thatinternalize virus or coat protein. The subject invention provides ahighly selective method of destroying HIV infected cells, utilizing theantibodies described herein.

While not wishing to be constrained to any particular theory ofoperation of the invention, it is believed that the expression of thetarget antigen on the infected cell surface is transient. The antibodiesmust be capable of reaching the site on the cell surface where theantigen resides and interacting with it. After the antibody complexeswith the antigen, endocytosis takes place carrying the toxin into thecell.

The immunotoxins of this invention are particularly helpful in killingmonocytes/macrophages infected with the HIV virus. In contrast to thetransient production of virus from T cells, macrophages produce highlevels of virus for long periods of time. Current therapy is ineffectivein inhibiting the production of new viruses in these cells.

Not all monoclonal antibodies specific for HIV env or unclipped HIV envmake highly cytotoxic immunotoxins, however assays are routinely andcommonly used in the field to predict the ability of an antibody tofunction as part of a immunotoxin. Preferably the antibodies used crossreact with several (or all) strains of HIV.

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or anenzymatically active toxin of bacterial, fungal, plant or animal origin,or an enzymatically active fragment of such a toxin. Enzymaticallyactive toxins and fragments thereof used are diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Inanother embodiment, the antibodies are conjugated to small moleculeanticancer drugs such as cis-platin or 5FU. Conjugates of the monoclonalantibody and such cytotoxic moieties are made using a variety ofbifunctional protein coupling agents. Examples of such reagents areSPDP, IT, bifunctional derivatives of imidoesters such as dimethyladipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-azido compounds such as his(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such as bis-(p-diazoniumbenzoyl)- -ethylenediamine, diisocyanates such as tolylene2,6-diisocyanate and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may bejoined to the Fab fragment of the antibodies.

Immunotoxins can be made in a variety of ways, as discussed herein.Commonly known crosslinking reagents can be used to yield stableconjugates.

Advantageously, monoclonal antibodies specifically binding the domain ofthe protein which is exposed on the infected cell surface, areconjugated to ricin A chain. Most advantageously the ricin A chain isdeglycosylated and produced through recombinant means. An advantageousmethod of making the ricin immunotoxin is described in Vitetta et al.,Science 238:1098 (1987) hereby incorporated by reference.

When used to kill infected human cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques.

Cytotoxic radiopharmaceuticals for treating infected cells may be madeby conjugating radioactive isotopes (e.g. I, Y, Pr) to the antibodies.Advantageously alpha particle-emitting isotopes are used. The term"cytotoxic moiety" as used herein is intended to include such isotopes.

In a preferred embodiment, ricin A chain is deglycosylated or producedwithout oligosaccharides, to decrease its clearance by irrelevantclearance mechanisms (e.g., the liver). In another embodiment, wholericin (A chain plus B chain) is conjugated to antibody if the galactosebinding property of B-chain can be blocked ("blocked ricin").

In a further embodiment toxin-conjugates are made with Fab or F(ab')₂fragments. Because of their relatively small size these fragments canbetter penetrate tissue to reach infected cells.

In another embodiment, fusogenic liposomes are filled with a cytotoxicdrug and the liposomes are coated with antibodies specifically bindingHIV env.

Antibody Dependent Cellular Cytotoxicity

The present invention also involves a method based on the use ofantibodies which are (a) directed against HIV env or unclipped HIV env,and (b) belong to a subclass or isotype that is capable of mediating thelysis of HIV virus infected cells to which the antibody molecule binds.More specifically, these antibodies should belong to a subclass orisotype that, upon complexing with cell surface proteins, activatesserum complement and/or mediates antibody dependent cellularcytotoxlcity (ADCC) by activating effector cells such as natural killercells or macrophages.

The present invention is also directed to the use of these antibodies,in their native form, for AIDS therapy. For example, IgG2a and IgG3mouse antibodies which bind HIV-associated cell surface antigens can beused in vitro for AIDS therapy. In fact, since HIV env is present oninfected monocytes and T-lymphocytes, the antibodies disclosed hereinand their therapeutic use have general applicability.

Biological activity of antibodies is known to be determined, to a largeextent, by the Fc region of the antibody molecule (Uananue andBenacerraf, Textbook of Immunology, 2nd Edition, Williams & Wilkins, p.218 (1984)). This includes their ability to activate complement and tomediate antibody-dependent cellular cytotoxicity (ADCC) as effected byleukocytes. Antibodies of different classes and subclasses differ inthis respect, and, according to the present invention, antibodies ofthose classes having the desired biological activity are selected. Forexample, mouse immunoglobulins of the IgG3 and IgG2a class are capableof activating serum complement upon binding to the target cells whichexpress the cognate antigen.

In general, antibodies of the IgG2a and IgG3 subclass and occasionallyIgG1 can mediate ADCC, and antibodies of the IgG3, IgG2a, and IgMsubclasses bind and activate serum complement. Complement activationgenerally requires the binding of at least two IgG molecules in closeproximity on the target cell. However, the binding of only one IgMmolecule activates serum complement.

The ability of any particular antibody to mediate lysis of the targetcell by complement activation and/or ADCC can be assayed. The cells ofinterest are grown and labeled in vitro; the antibody is added to thecell culture in combination with either serum complement or immune cellswhich may be activated by the antigen antibody complexes. Cytolysis ofthe target cells is detected by the release of label from the lysedcells. In fact, antibodies can be screened using the patient's own serumas a source of complement and/or immune cells. The antibody that iscapable of activating complement or mediating ADCC in the in vitro testcan then be used therapeutically in that particular patient.

Antibodies of virtually any origin can be used for this purpose providedthey bind a HIV env epitope and can activate complement or mediate ADCC.Monoclonal antibodies offer the advantage of a continuous, ample supply.In fact, by immunizing mice with gp160 establishing hybridomas makingantibodies to HIV env and selecting hybridomas making antibodies whichcan lyse infected cells in the presence of human complement, it ispossible to rapidly establish a panel of antibodies capable of reactingwith and lysing infected cells.

Therapeutic and Other Uses Of the Antibodies

When used in vivo for therapy, the antibodies of the subject inventionare administered to the patient in therapeutically effective amounts(i.e. amounts that restore T cell counts). They will normally beadministered parenterally. The dose and dosage regimen will depend uponthe degree of the infection, the characteristics of the particularimmunotoxin (when used), e.g., its therapeutic index, the patient, andthe patient's history. Advantageously the immunotoxin is administeredcontinuously over a period of 1-2 weeks, intravenously to treat cells inthe vasculature and subcutaneously and intraperitoneally to treatregional lymph nodes. Optionally, the administration is made during thecourse of adjunct therapy such as combined cycles of tumor necrosisfactor and interferon or other immunomodulatory agent.

For parenteral administration the antibodies will be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic, and non-therapeutic. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate can also be used. Liposomes may be used as carriers. The vehiclemay contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies will typically be formulated in such vehicles atconcentrations of about 1 mg/ml to 10 mg/ml.

Use of IgM antibodies is not currently preferred, since the antigen ishighly specific for the target cells and rarely occurs on normal cells.IgG molecules by being smaller may be more able than IgM molecules tolocalize to infected cells.

There is evidence that complement activation in vivo leads to a varietyof biological effects, including the induction of an inflammatoryresponse and the activation of macrophages (Uananue and Benecerraf,Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).The increased vasodilation accompanying inflammation may increase theability of various anti-AIDS agents to localize in infected cells.Therefore, antigen-antibody combinations of the type specified by thisinvention can be used therapeutically in many ways. Additionally,purified antigens (Hakomori, Ann. Rev. Immunol. 2:103 (1984)) oranti-idiotypic antibodies (Nepom et al., Proc. Natl. Acad. Sci. 81:2864(1985); Koprowski et al., Proc. Natl. Acad. Sci. 81:216 (1984)) relatingto such antigens could be used to induce an active immune response inhuman patients. Such a response includes the formation of antibodiescapable of activating human complement and mediating ADCC and by suchmechanisms cause infected cell destruction.

The antibodies of the subject invention are also useful in the diagnosisof HIV in test samples. They are employed as one axis of a sandwichassay for HIV env or unclipped HIV env, together with a polyclonal ormonoclonal antibody directed at another sterically-free epitope of HIVenv of unclipped HIV env. For use in some embodiments of sandwich assaysthe 10F6, 11G5, or 10D8 antibody or its equivalent is bound to aninsolubilizing support or is labelled with a detectable moiety followingconventional procedures used with other monoclonal antibodies. Inanother embodiment a labelled antibody, e.g. labelled goat anti-murineIgG, capable of binding the 10F6, 11G5, or 10D8 antibody is employed todetect HIV env or unclipped HIV env binding using procedures previouslyknown per se.

The antibodies of this invention which are directed to an epitopespanning the clip site are used for producing unclipped HIV env. Thismethod comprises the following general steps: First, contacting a firstpreparation of HIV env with an antibody directed to an HIV env epitopespanning the clip site for a time sufficient to permit formation of asecond, antibody-bound HIV env, preparation; secondly, separating thesecond preparation from any HIV env which is not antibody-bound; andthirdly recovering the unclipped HIV env from said second preparation.These methods are used with standard procedures for affinitypurification and affinity chromatography, and may use antibody which isimmobilized on a water insoluble support as discussed above. Any knownprocedure for immobilizing monoclonal antibodies can be used for thispurpose.

The antibody compositions used in therapy are formulated and dosagesestablished in a fashion consistent with good medical practice takinginto account the disorder to be treated, the condition of the individualpatient, the site of delivery of the composition, the method ofadministration and other factors known to practitioners. The antibodycompositions are prepared for administration according to thedescription of preparation of polypeptides for administration, infra.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

In particular, it is preferred that these plasmids have some or all ofthe following characteristics: (1) possess a minimal number ofhost-organism sequences; (2) be stable in the desired host; (3) becapable of being present in a high copy number in the desired host; (4)possess a regulatable promoter; and (5) have at least one DNA sequencecoding for a selectable trait present on a portion of the plasmidseparate from that where the novel DNA sequence will be inserted.Alteration of plasmids to meet the above criteria are easily performedby those of ordinary skill in the art in light of the availableliterature and the teachings herein. It is to be understood thatadditional cloning vectors may now exist or will be discovered whichhave the above-identified properties and are therefore suitable for usein the present invention and these vectors are also contemplated asbeing within the scope of this invention.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.8:4057 (1980).

"PCR" (polymerase chain reaction) refers to a technique whereby a pieceof DNA is amplified. Oligonucleotide primers which correspond to the 3'and 5' ends (sense or antisense strand-check) of the segment of the DNAto be amplified are hybridized under appropriate conditions and theenzyme Taq polymerase, or equivalent enzyme, is used to synthesizecopies of the DNA located between the primers.

"Dephosphorylation" refers to the removal of the terminal 5' phosphatesby treatment with bacterial alkaline phosphatase (BAP). This procedureprevents the two restriction cleaved ends of a DNA fragment from"circularizing" or forming a closed loop that would impede insertion ofanother DNA fragment at the restriction site. Procedures and reagentsfor dephosphorylation are conventional. Maniatis, T. et al., MolecularCloning pp. 133-134 (1982). Reactions using BAP are carried out in 50 mMTris at 68° C. to suppress the activity of any exonucleases which arepresent in the enzyme preparations. Reactions are run for 1 hour.Following the reaction the DNA fragment is gel purified.

"Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinage. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T. et al., Id., p.146). Unless otherwise provided, ligation is accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5μg of approximately equimolar mounts of the DNA fragments to be ligated.

"Filling" or "blunting" refers to the procedures by which the singlestranded end in the cohesive terminus of a restriction enzyme-cleavednucleic acid is converted to a double strand. This eliminates thecohesive terminus and forms a blunt end. This process is a versatiletool for converting a restriction cut end that may be cohesive with theends created by only one or a few other restriction enzymes into aterminus compatible with any blunt-cutting restriction endonuclease orother filled cohesive terminus. Typically, blunting is accomplished byincubating 2-15 μg of the target DNA in 10 mm MgCl₂, 1 mMdithiothreitol, 50 mM NaCl, 10 mM Trig (pH 7.5) buffer at about 37° C.in the presence of 8 units of the Klenow fragment of DNA polymerage Iand 250 μM of each of the four deoxynucleoside triphosphates. Theincubation generally is terminated after 30 min. phenol and chloroformextraction and ethanol precipitation.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture appear below, but should not be construed to limit theinvention. All literature citations herein are expressly incorporated byreference.

Deposit of Materials

The following cultures have been deposited with the American TypeCulture Collection, 12301 Parklawn Drive, Rockville, Md., USA(ATCC):______________________________________Strain ATCC Dep. No.Deposit Date______________________________________gp120 in -- --6E10 CRL10514 26 JULY 19905B3 CRL 10515 26 JULY 199013H8 CRL 10510 26 JULY199010F6 CRL 10512 26 JULY 199011G5 CRL 10511 26 JULY 19906D8 -- --10D8CRL 10513 26 JULY 1990.______________________________________

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of viable cultures for 30 years fromthe date of the deposit. The organisms will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the cultures to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.12 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureson deposit should die or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the constructs deposited,since the deposited embodiments are intended to illustrate only certainaspects of the invention and any constructs that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial herein does not constitute an admission that the writtendescription herein contained is inadequate to enable the practice of anyaspect of the invention, including the best mode thereof, nor is it tobe construed as limiting the scope of the claims to the specificillustrations that they represent. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

EXAMPLES Example 1 CHIMPANZEE VACCINE STUDY

Previous literature reported that chimpanzees immunized with recombinantgp120 (rgp120) developed humoral and cellular immunity to virus-derivedprotein, but that vaccination with this preparation failed to provideprotection from HIV-1 infection in vivo (e.g. Berman et el., Proc. Nat.Acad. Sci. USA 85:5200-5204, 1988 and other references, infra.Surprisingly, our present study demonstrates a vaccine which did provideprotection.

The proteins used in this study were purified preparations ofrecombinant protein expressed in mammalian cell culture and adsorbedonto aluminum gel.

The rgp120 protein (FIG. 3A) consists of the gp120 fragment of the HIV-1envelope glycoprotein fused to a short N-terminal sequence of the herpessimplex virus glycoprotein D to facilitate expression. The sgp160protein is a variant of gp1 60 wherein the transmembrane domain andcytoplasmic tall have been deleted to enable the protein to be secretedfrom mammalian cells. Both proteins are glycosylated in a manner similarto authentic viral gp120, and bind to CD4, the cellular receptor forHIV-1, with high affinity.

The immunogens used in this study, rgp120 and sgp160, were purified byimmunoaffinity chromatography using different monoclonal antibodies foreach antigen, as described above. Besides the absence of contaminatingproteins, a major difference between the gp120 used in this study andprevious studies is that the present study used gp120 which was notproteolyzed. Evaluation of material used in previous studies shows thatapproximately 50% of the gp120 in recombinant preparations wasproteolytically clipped at amino acid 315 to yield peptides thatmigrated on SDS PAGE gels with mobilities of 50 kd and 75 kd. We havedetermined that this proteolysis site is located in the middle of amajor type-specific neutralizing epitope (shown as IV on FIG. 1). Inthis study, we used unclipped gp120, but the sgp160 preparation usedexhibited approximately 50 % proteolysis at this position.

In the present study, two chimpanzees were immunized with the 140-150 kdsgp160 protein, and two chimpanzees were immunized with the 120-130 kDrgp120 protein. A placebo control animal was immunized with the samequantity of recombinant glycoprotein D (gD-1) of herpes simplex virustype 1, expressed in mammalian cell culture, purified by a similarprocedure as the rgp120 (Berman et al., Science 277:1490-1492 (1985)),and adsorbed onto aluminum gel. Specifically, animals were immunizedwith 1 ml of a preparation of antigen at a dose of 300 μg of protein peranimal per immunization.

All antigens were formulated in an aluminum hydroxide (alum) adjuvant(Berman et al., Proc. Natl. Acad. Sci. USA 85:5200-5204 (1988) (0.5mg/ml of Al⁺³) in phosphate buffered saline (0.016M PO₄ pH 6.2, 0.15MNaCl, 0.004M KCl), the only adjuvant currently approved in the UnitedStates for vaccine products.

The immunization schedule that we employed was different from allpublished HIV or SIV vaccine trials, but was similar to that used forthe Hepatitis B virus vaccine which was found to give good results indose interval studies in other primates (Anderson et al., J. Infect.Diseases 160:960-969 (1989). Animals were given a primary immunizationwith either unclipped rgp120 or sgp160 at time 0, followed by boosterimmunizations at four weeks and thirty-two weeks. Test and controlpreparations were administered intramuscularly in two sites (0.5ml/site).

Blood samples were taken from each animal at bimonthly intervals andanalyzed by a number of assays. Besides immunological assays (e.g.antibody titers to gp120, lymphocytes proliferation assays), the levelof T cell subsets, serum enzyme levels, and clinical chemistries weremonitored. As reported previously, we found no abnormalities inlymphocyte subsets, lymphocyte function, serum enzyme levels, or bloodchemistries resulting from immunization with these proteins. Thusconcerns that immunization with rgp120 or sgp160 might lead to untowardside effects (e.g. immunosuppression or autoimmunity) could not bevalidated by these studies.

After the primary immunization, a low level of antibodies to sgp 160 butnot rgp120 were detected by commercial (Dupont or Biorad) HIV-1immunoblot assays, but not (FIG. 6A) in a liquid-phaseradioimmunoprecipitation assay (RIA) or a commercial (Genetic Systems)HIV-1 antibody test (ELBA). A second immunization at four weeks eliciteda moderate humoral and cellular immune response to both of the HIV-1derived immunogens that could be detected in a liquid phaseradioimmunoprecipitation assay (RIP) (FIG. 6A). and immunoblot assays(data not shown), but not by ELISA. The third immunization, given at 32weeks (seven months following the second) resulted in a marked increasein antibody titers apparent in all three antibody assays (FIGS. 6 and 7.Immunoblot analysis (FIG. 7) of the serum collected three weeks afterthe last boost (week 35) showed that those that received rgp120 reactedstrongly with HIV-1 gp120 and to a lesser extent with gp160. Animalsimmunized with rgp160 reacted, as intended, with viral gp160, and gp41as well as gp120. Also evident in immunoblots were antibodies reactivewith proteolytic breakdown products of gp120. Cellular immunity, asindicated by the ability of rgp120 to stimulate lymphocyteproliferation, could be detected at this time in the rgp120 and sgp160immunized animals but not in the control (Table 2).

The increase in antibody titers to rgp120 and sgp160 observed after thethird immunization was coincident with the appearance of neutralizingantibodies (Robertson, et al., J. Virol. Methods. 20:195-202 (1988))(FIG. 8A). The time required to attain the peak neutralizing titervaried from two to four weeks after boosting, however no significantdifference in the magnitude of the peak in vitro neutralizing titers wasdetected between those animals immunized with rgp120 and with sgp160.One animal from each group exhibited peak neutralizing titers of 1:640whereas the remaining animals in each group possessed peak titers of1:320. Significantly, these neutralizing responses were far greater thanthose observed previously (Berman et al., 1988, supra) where theneutralizing activity of sera to rgp120 could only be detected at a 1:5dilution in a neutralizing assay with comparable sensitivity. Thus, theimmunogens and immunization procedures used in the present studyelicited a neutralizing response that was 1-2 orders of magnitudegreater than that previously observed.

Based on the significant levels of neutralizing antibodies, we proceededwith a virus challenge of these animals. All five animals wereintravenously injected, three weeks after the final immunization (week35), with approximately 40 tissue culture infectious dose units (TCID₅₀)of virus derived from a standard inoculum of the IIIB isolate of HIV-1(kindly provided by Dr. L. Arthur, National Cancer Institute, NationalInstitute of Health, Bethesda Md., USA) that has been used for manyother chimpanzee infectivity studies (Arthur et al., supra, and Princeet al., supra). The envelope protein from this isolate is 98% identicalin amino acid sequence with that used to produce the rgp120 and sgp160immunogens, described above. The amount of virus was used in this studywas the same for all challenged animals and corresponded toapproximately ten chimpanzee infectious doses (CID₅₀) Blood was takenfrom the animals at bimonthly interval and was subjected to a battery ofassays to detect vital infection.

After virus challenge, the magnitude and the duration of the anti-rgp120antibody response in the rgp120 immunized animals differed markedly fromthe other animals in teh study. The titer of anti-gp120 antibodies inthese animals as measured by RIA did not increase after virus challenge,but rather, progressively declined to baseline values (Table 2). Incontrast, the titer of anti-gp120 antibodies in the animals immunizedwith sgp160 modestly declined for approximately 7-8 weeks after viruschallenge, then increased to a titer somewhat higher than the peakachieved by hyperimmunization, and then leveled off at an intermediatetiter. These results suggested that HIV-1 challenge of teh sgp160immunized animals resulted in viral infection and the production ofsufficient vital protein to stimulate an anamnestic immune response. Theobservation that the titer of antibodies to gp120 in thergp120-immunized animals did not increase after challenge, but in factdisappeared with time, suggests that a productive infection did notoccur. Antibodies reactive with rgp120 were detected in the controlanimal by RIA at 16 weeks post challenge, indicating that this animalalso became infected with HIV-1.

Analysis of these sen by ELISA using commercially available HIV-1antibody assay (FIG. 6B) showed a pattern of activity that differed formthat detected in the anti-gp120 RIA. In this assay, antibodies to HIV-1were detected only after the third immunization, and the antibodyresponse present was significantly greater in the rgp160-immunizedanimals than in the rgp120 animals. This difference reflects the factthat the commercial HIV-1 antibody assays typically possess less gp120than gp41. Sera from the sgp160-immunized animals gave a strong reactionin this assay because they contained antibodies to gp41 as well asgp120. The control animal showed no signs of HIV-1 reactive antibodiesat any time prior to HIV-1 challenge. The HIV-1 antibody titers in theanimals immunized with rgp160 declines for approximately 6-7 weeks aftervirus challenge, and then increased to a level similar to that obtainedby hyperimmunization and then declined to intermediate values. Incontrast, ELISA titers in teh rgp 120-immunized animals did notincrease, but rather progressively declined to baseline values by 7Weeks post challenge (Table 2, FIG. 6B). This result furtherdemonstrated that there was not sufficient replication of HIV-1 in thergp120-immunized animals to elicit an immune response to any of theviral antigens contained in the HIV-1 antibody assay. The control animalwas negative in this assay at the time of challenge, but became positivein response to viral infection at 13 weeks post challenge, indicatingthat this animal became infected with HIV-1.

Immunoblot analysis of chimpanzee sera showed that at the time ofchallenge the sera from the rgp120 and sgp160-immunized animalspossessed antibodies to the HIV-1 envelope glycoproteins, but not to thecore proteins (e.g. p17, p24, and p55). Serologic evidence of HIV-1infection in the two sgp160-immunized animals was first noted at 5 weekspost challenge where a faint p24 band appeared (data not shown).Antibodies to p17 and p55 became apparent at later dates (Table 2, FIG.7). Antibodies to p24 were first detected in the sera of the controlanimal at 9 weeks post challenge (data not shown), and reactivity withp17, p55 and gp120 could be detected at later dates. Interestingly, theintensity of the antibody response to HIV-1 structural proteins (e.g.p55, p24 and p17) was far greater in the sgp160-immunized animals (FIG.7) than in the control. Antibodies to gp120 were present in thergp120-immunized animals at the time of challenge, but had largelydisappeared by 13 weeks post challenge (FIG. 7). Significantly, theanimals immunized with rgp120 have not, as of 26 weeks post challenge,seroconverted to any of the HIV-1 encoded proteins other than gp120,suggesting that they were resistant to HIV-1 infection.

Changes in the concentration of neutralizing antibodies after HIV-1challenge paralleled those seen in the anti-gp120 titers (FIGS. 6A and7). Thus, the 32-week immunization elicited a high level of neutralizingantibodies in the rgp120 and sgp160-immunized animals which persistedthrough the time of HIV-1 challenge at 35 weeks (Table 2). Theneutralizing titers in the rgp120-immunized animals reached peak titersby the time of virus challenge and the n steadily wanted to the level ofpre-immune sera by 60 weeks. The neutralizing titers in both of thesgp160-immunized animals declined for several weeks but sharplyincreased at 7 weeks post-challenge, presumably in response to theproduction of viral proteins. Neutralizing titers in one of the sgp 160animals rose to values of 1:1280 by 13 weeks post challenge.Neutralizing antibodies were not detected in the control until 17 weekspost challenge. The results of these assays provided further evidencethat HIV-1 did not replicate in the rgp120-immunized animals but did soin the control and the sgp160-immunized animals.

Virus co-cultivation studies were carried out in order to determinewhether viable virus could be recovered from any of the immunizedanimals. HIV-1 could be recovered from the control and thesgp160-immunized animals at 6-7 weeks post challenge (Table 2) but hasnever been recovered from either of the rgp120-immunized animals at anytime up to 6 months post challenge. Further evidence of HIV-1 infectionin the control and the two sgp160-immunized animals was provided by PCRanalysis (Table 2) where proviral DNA could be detected in theseanimals, but not the rgp120-immunized animals. Taken together, theseresults suggest that immunization with rgp120 elicited a protectiveimmune response which has significantly delayed or completely preventedinfection of these animals by HIV-1.

Since the levels of neutralizing antibodies present in the rgp120 andsgp160-immunized chimps were essentially identical over the four-weekinterval that followed the last boost, it was surprising that only thergp120-immunized animals were protected from HIV-1 infection. Oneexplanation for the difference in susceptibility to HIV-1 infectionbetween the two groups of animals is that on the actual day of viruschallenge, the sgp160--immunized animals had slightly lowerneutralization titers (i.e. 1:160) than the rgp120-immunized animalswhich exhibited neutralization titers of 1:320 and 1:640. If the levelof antibody present on the actual day of challenge is of overridingimportance, than our data suggest that titers of >1:160 are required toprevent infection. On balance, we considered it unlikely that there issuch a discrete threshold in protection from infection and postulatedthat other factors may be involved.

Additional studies were carried out to determine whether antibodies tospecific epitopes of functional significance might better correlate withprotection. We reasoned that antibodies able to block the binding ofgp120 to CD4 might play an important role. Previously, (Berman et al.,J. Virol. 63:3489-3498 (1989); Lasky et al., Cell 50:975-985 (1987)) wenoted that rgp120 and sgp160 were both effective in eliciting antibodiesthat disrupted this interaction. Analysis of sera at the time ofchallenge showed that all the animals with the HIV-1 derived immunogenspossessed antibodies that blocked CD4 binding, and that the blockingtiter did not correlate with protection from infection or withneutralizing titer (Table 2). Another possibility we considered waswhether a subpopulation of neutralizing antibodies might account for thedifference in protection between the rgp120 and wgp160 treated animals.In these studies we assayed the sera for antibodies reactive with apeptide within the V3 domain of gD120 known to contain the majortype-specific neutralizing determinant (MND) (Matsushita et al., J.Virol. 62:2104-2114 (1988)).

Antibodies to this epitope were detected using an ELISA assayincorporating a synthetic MND peptide, RP135, described by Matsushita,ibid. When sera form the rgp120 and sgp160-immunized animals werecompared, we found that antibody binding to the MND peptide differedsignificantly between the two groups of animals (Table 2, FIG. 8B). Atthe time of challenge, there was almost a ten-fold difference in theamount of antibodies to the MND in the animals immunized with rgp120,compared to those immunized with sgp160. Especially interesting was theobservation that one animal, which had the lowest anti-gp120 titer byRIA, had the highest titer to the MND, and was protected from infection.Thus, the correlation between protection and the level of antibodies tothe MND appeared to be stronger than the correlation between protectionand the level of antibodies to the MND appeared to be stronger than thecorrelation between neutralizing antibodies and protection.

The difference in the formation of antibodies to the MND may beattributable to the difference the amount of proteolytic processing(FIG. 3) between the two preparations (i.e. 40% for sgp160 and 5% forrgp120) especially since the clip site, in this instance arginineresidue 312, is located within a disulfide bonded loop that contains theMND. It should be noted that proteolysis at this position does notappear to cause a major conformational change in either rgp120 or sgp160since proteolyzed material is able to bind to CD4 with high affinity(Berman and Gregory, unpublished results). As with the RIA titers andthe HIV-1 antibody titers, we found that the concentration of antibodiesto this epitope reached maximum values in the rgp120 animals shortlyafter the final immunization (32 weeks) and then progressively declinedto baseline values. Antibodies from the sgp160 immunized animals peakedshortly after the last injection and proceeded to decline for severalweeks, then increased and have remained positive at more than 25 weekspost challenge. Antibodies to the MND appeared in the control animal at15 weeks post challenge.

Another significant result of these studies is that there are at leasttwo populations of antibodies able to neutralize HIV-1 infectivity invitro. One population is elicited to rgp120 and is presumably directedtowards the MND. A second population is elicited by immunization withrsgp 160 and is directed towards sites other than the MND. While thehypothesis that in vivo protection is dependent on the presence ofantibodies to the MND is supported by the data for the rgp120 immunizedanimals, we cannot be certain that the lack of protection in the sgp160immunized animals was due to a low level of antibodies to the MND. Analternative explanation, that would also fit our data, is that theneutralizing antibodies elicited by sgp160 to sites other than the MNDare, in fact, able to provide protection in vivo, but that some factorinterferes with or abrogates their protective effect. There have beenseveral reports of antibodies to HIV-1 that enhance rather than inhibitviral infection in vitro (Homsy et al., Science 244:1357-1359 (1989);Robinson et al., Lancet 1:790-794 (1988). An enhancing epitope containedon sgp160, but not rgp120, might account for the difference inprotection that we have observed. This possibility would be consistentwith the observation that the animals immunized with sgp160 (x-247 andx-261) seroconverted to HIV-1 core proteins earlier (5 weeks postchallenge) than the control (x-246) which seroconverted at 11 weeks postchallenge (Table 2), and that the intensity of the immune response, asvisualized in the immunoblot assay, was much greater for these animalthat for the control (FIG. 7). Recently, two different enhancingepitopes have been mapped to a region of gp41 (Robinson et al., Proc.Natl. Acad. Sci. USA (in press))that was present on the sgp160immunogen, but not the rgp120 immunogen.

Several factors may account for the success of rgp120 in the currentstudy as compared to our previous study (Berman et al., supra) wherergp120 failed to provide protection from HIV-1 infection. Onesignificant difference was that the animals were immunized according toan optimized immunization protocol, whereas previously an immunizationprotocol that had been adapted from rodent studies was employed. Anotherimportant difference was that the rgp120 used in the earlier study wasonly 50% pure and was approximately 50% clipped, whereas the rgp120employed in the present studies was <5% clipped (FIG. 3). A thirddifference was that in the previous study the animals were challengedwith 100 TCID₅₀ units (25 CID₅₀) of HIV-1, whereas in the present studythe animals were challenged with 40 TCID50 units of virus (10 CID₅₀). Afinal explanation for the difference between the present study and theprevious study relates to the experimental design. Because theavailability of chimpanzees restricts the size of the treatment groupsto only a few animals, the ability to show statistical significance inchimpanzee efficacy studies is limited. Thus, negative results areuninterpretable and do not allow us to discern whether the rgp120 usedin the previous studies, or the sgp160 employed in the present study,did or did not confer some significant level of protective immunity.

This application represents the first report, to our knowledge, of acandidate AIDS vaccine that has succeeded in protecting chimpanzees fromHIV-1 infection. Significantly, the recombinant subunit vaccinedescribed herein consists of a single purified protein, preparedentirely from non-infectious materials, and is effective in an adjuvantapproved for human use.

                                      TABLE 2                                     __________________________________________________________________________    Immunologic and Virologic Characteristic Of                                   Chimpanzees Before and After HIV-1 Infection*                                               CONTROL                                                                             SGP160   RGP120                                                         x246  x247                                                                              x261 x262                                                                              x265                                         __________________________________________________________________________    I. Characteristics at the time of virus challenge (35 weeks)                    Anti-gp120 Titer                                                                          -     4.3 4.3  4.8 3.9                                            Antibodies to gp41                                                                        -     +   +    -   -                                              HIV-1 ELISA Titer                                                                         <2400 6400                                                                              25,600                                                                             3200                                                                              400                                            Proliferation to rgp120                                                                   -     +   +    +   +                                              Neutralizing Titer                                                                        <10   160 160  320 640                                            CD4 Blocking Antibodies                                                                   <1    2.2 2.5  2.5 1.7                                            Anti-MND Titer                                                                            <10   145 149  491 844                                          II. Characteristics After HIV-1 Challenge                                       1ST Cocultivation of HIV-1                                                                6     6   7    -   -                                              Time to anti-p24 (wk)                                                                     11    5   5    -   -                                              Time to p17 (wk)                                                                          13    5   5    -   -                                              PCR (53 wk) +     +   +    -   -                                              HIV-1 ELISA Titer (60 wk)                                                                 800   3200                                                                              6400 <400                                                                              <400                                           Neutralizing Titer (60 wk)                                                                10    80  80   <10 <10                                            Proliferation to rgp120                                                                   -     +   +    -   -                                              (60 wk)                                                                       Anti-gp120 Titer (63 wk)                                                                  -     3.2 2.9  -   -                                            __________________________________________________________________________     *Legend to Table 2: Antibody titers to gp120 were determined in a liquid      phase RIA; the data presented represent log values from endpoint dilution     titrations (see description of FIG. 6A). The presence of antibodies to        gp41 and the time to appearance of antibodies to p24 and p17 were             determined by immunoblot analysis of sequential bleeds using commercial       (Dupont and Biorad) Western blot strips. Antibodies that block the bindin     of gp120 to CD4 were measured as described previously, data represents th     log of endpoint dilution titrations. HIV1 ELISA titers were determined in     a commercial HIV1 antibody assay kit (see description of FIG. 6B). The        cocultivation of HIV1 and the proliferation of chimpanzee lymphocytes to      rgp120 was carried out as described previously. In vitro neutralizing         antibodies were measured by the method of Robertson, et al.. supra. (see      description to FIG. 8). AntiMND titers were determined in an ELISA assay      as described above. PCR reactivity was determined using the method of         Kellog and Kwok, in PCR Protocols (Innis et al., eds) 337-347 (Academic       Press, New York, 1990). Briefly, frozen lymphocytes were processed in PCR     lysis buffer and the DNA was phenolchloroform extracted and ethanol           precipitated. A sample of the DNA (equivalent to the amount in                150,000-300,000 cells) was subjected to 40 cycles of PCR using the SK68       and SK69 primers. Samples were adjusted to 25 mM NaCl and hybridized in       solution for 30 min at 55° C. to 0.5 pmol of primer SK70 end           labeled with gamma labeled  .sup.32 P!- ATP. Samples were resolved on a       10% acrylamide minigel in TBE buffer. The gel was then dried and used to      expose an xray film. Positive control DNA was prepared from a transfected     CHO cell line expressing gp160. DNA samples were tested for their ability     to be amplified using probes to beta globin. Sample were considered           positive for HIV1 if the labeled primer hybridized to a band of 141 base      pairs.                                                                   

Example 2 GENERATION OF ANTIBODIES

1. Generation and Characterization of Monoclonal Antibodies.

Soluble forms of recombinant gp120 and gp160 were expressed in ChineseHamster Ovary (CHO) cells according to the methods described in Lasky etal., Science 223:209 (1986), and were purified by affinitychromatography from growth conditioned cell culture medium of the D531and D683.DC.9 cell lines described previously. The soluble form ofrecombinant gp160 (sgp160 or 683DC.7) contained two deletions. The firsteliminated ten amino acids spanning the gp120/41 cleavage site and thesecond deleted the hydrophobic transmembrane and cytoplasmic tail.

For ELISA assays a gp41 fusion protein (LE41 ) consisting of an aminoterminal fragment of the Trp E gene fused to 100 amino acids from theamino terminus of gp41 was expressed in E. coli and purified aspreviously described. Reduced and carboxymethylated rgp120 and sgp160were prepared by dialysis of sgp160 and rgp120 into Tris/HCl buffercontaining urea and EDTA. Dithiothreitol (DTT) was added to yield 10 mMfinal volume, and the proteins were mixed four hours at roomtemperature. Iodoacetic acid was added to 25 mM final volume, and thesamples were dialyzed into ammonium bicarbonate buffer. These proteinswere used to coat ELISA plates at 1.0 μg/ml, and the purifiedmonoclonals screened in a standard ELISA protocol.

Both soluble forms of the HIV env protein were affinity purified andthen used to immunize commercially available Balb/c mice for theproduction of monoclonal antibodies (MAbs). Each mouse was immunizedwith 20 μg of rgp120 or sgp160 (683ΔC.7) intraperitoneally (i.p.) orintravenously (i.v.). and boosted three days prior to fusion. The mousewith the highest antisera titer was selected for fusion. The mousemyeloma line NP3x63-Agg.653 was fused with spleen cells in a 4:1 ratiousing 50% polyethylene glycol, although other commercially availablemyeloma lines may be utilized according to established procedures.Hybrids were selected for growth with media supplemented withhypoxanthine and azaserine. Positive parental supernatants wereidentified by screening individual wells against rgp120 or sgp160 in asolid-phase enzyme-linked immunosorbent assay (ELISA). Reactive wellswere expanded, cloned by limiting dilution, and the hybridoma cellsinjected into pristine-primed Balb/c mice. Ascites fluid was collected,pooled, and purified by protein-A column chromatography.

Ten stable monoclonal antibody producing cell lines against 683DC.7 andeleven cell lines were produced over several fusions. They had beeninitially screened by their ability to bind recombinant gp120/gp160 insolid phase ELISAs. Seventeen of the MAbs reacted with gp120, while theremaining reacted with the gp41 portion of the molecule. Solid phaseELISAs were performed as follows: ELISA microtiter plates were coatedwith recombinant sgp160, rgp120, or LE41 at 1.0 μg/ml. After blockingwith 0.5% BSA/PBS (bovine serum albumin/phosphate-buffered saline), 100μl of purified antibody was tested at a concentration of 10 μg/mlfollowing a standard ELISA procedure.

Subsequent assays were performed to titrate relative strength of bindingof gp120 to CD4, and to determine relative affinities. Purified antibodywas titered against rgp120 or sgp160, and the half maximum O.D. wascalculated from a titration curve (results not shown).

Additionally, an antigen capture assay was used to select higheraffinity antibodies which bind soluble antigen. Dynatech polystyreneremovable strip wells were coated with 0.5 μg goat anti-mouse IgG andblocked with 0.5% BSA/PBS. After washing, 100 μl of purified antibody at10 μg/ml was added and incubated for two hours at 37° C. The strips werewashed and labelled with I¹²⁵ -683ΔC.7, 10⁶ c.p.m. per well, for twohours at room temperature. Finally, the wells were washed, broken apartand counted on a gamma counter (results not shown).

Nineteen of the MAbs were of the IgG1 isotype while the remaining twowere IgG2a. Isotypes were determined by MonoAb-ID EIA kit available fromZymed (South San Francisco, Calif., USA) according to the vendor'sprotocol for isotype determination. Plates were coated with gp160 at 1.0μg/ml.

2. Epitope Mapping of Monoclonal Antibodies.

The MAbs described above define approximately eleven epitopes on gp160.In general, various epitope mapping methods were consistent in theirresults. Based on western data, ELISA assays with reduced andcarboxymethylated (RCM) gp160/120, and lambda gt11 mapping, the majorityof the antibodies appear to bind linear epitopes. One antibody, 6E10,appears to bind to a conformational epitope although it does retain somereactivity on western blot analysis. Methods and results are as follows.

1. Western Immunoblots: Naturally occurring proteolytic cleavage ofgp120 resolves three major polypeptides in a 7.5% polyacrylamide SDSgel. Separated are a 75,000 dalton N-terminal and a 55,000 daltonC-terminal band which comprise gp120. Further cleavage within the 55,000dalton C-terminal band yields a 35,000 dalton COOH-terminal band.Western blot analysis of the monoclonal antibodies with theseproteolytic cleavage fragments of gp120 was useful in the initialmapping of the epitopes.

2. Epitope Cross-Competition ELISA: The MAbs were tested for theirability to compete for epitope binding sites on both the gp120 and 160proteins. ELISA microliter plates were coated with sgp160 or rgp120 at1.0 μg/ml After blocking with BSA/PBS, 50 μl of purified MAb at 100μg/ml was added and incubated one hour at room temperature. Withoutwashing, 50 μl of each antibody coupled to horseradish-peroxidase wasadded for an additional hour. The plate was washed, substrate added, andread to 490 nm.

3. Lambda gt11 Mapping. A library was constructed containing randomlyexpressed portions of the gp160 precursor fused to beta galactosidase.Briefly, the 3.5 kb HTLV-IIIB gp160 region was treated with increasingquantities of DNAse I (described in copending U.S. Ser. No. 07/448038),blunted with the Klenow fragment of DNA polymerage and 4 deoxynucleotidetriphosphates, and ligated to EcoRI oligonucleotide linkers. Thematerial was run on a 5% acrylamide gel, and the 100-1100 bp region ofthe gel was isolated. DNA in this region was eluted and ligated to 1 μgof lambda gt11 EcoRl-cut phage arms. The ligated DNA was packaged invitro and amplified in E. coli 1088. The library consisted of 1.6×10⁷independent phage.

The library was screened by plating approximately 4×10³ phage onto E.coli Y1090 cells. After plaques developed, a nitrocellulose filterimpregnated with isopropylthiogalactoside was placed onto the plate andincubated overnight. The filters were blocked and incubated with MAb.Colorimetric detection of the antibody was achieved using horseradishperoxidase conjugated with goat anti-mouse antibody.

Positively reacting phage were plaque purified, and the DNA wasisolated. Double-stranded lambda DNA sequencing was carried out at 50°C. with klenow DNA polymerage and dideoxynucelotides in the presence ofthe following beta galactosidase gene specific oligonucleotide primers:5'-TTGACACCAGACCAACTGGTAATG-3'(reverse or carboxy- terminus) and5'-ATGGGGATTGGTGGCGACTCCTGGAGCCCG-3' (forward or amino-terminus). Theobserved DNA sequence resulted in the coordinates of the inserted gp120fragment.

4. Peptides: Peptides from various regions of gp120 were eithersynthesized or isolated by affinity purification of various digests. Thepeptides were spotted onto nitrocellulose and then reacted with thevarious monoclonal antibodies. Goat anti-mouse IgG conjugated tohorseradish peroxidase or iodinated Protein A was used to probe forreactivity of monoclonal antibodies with the peptides.

5. RCM 160/120: The MAbs were screened by ELISA against reducedcarboxymethylated (RCM) gp160/120 to determine if they reacted withlinear or conformational epitopes. 683ΔC.7 and rgp120 were dialyzed intoTris/HCl containing urea and EDTA. The following day, dithiothreitol(DTT) was added to yield 10 mM final volume, and the proteins were mixedfour hours at room temperature. Iodoacetic acid was added to 25 mM finalvolume and the samples were mixed 30 minutes in the dark. DTT was addedto yield 100 mM final volume, and the samples were dialyzed intoammonium bicarbonate. The proteins were used to coat ELISA plates at 1.0μg/ml, and the MAbs screened in a standard ELISA protocol.

Results of the epitope mapping are summarized in Table 3.

                  TABLE 3*                                                        ______________________________________                                                                 Lambda         RCM                                   MAb   Western   Epitope  gt11   Peptides                                                                              160/120                               ______________________________________                                        1D10  120k, 75k G        a.a. 65-85     +                                     1F9   120k, 75k L        233-274        +                                     5B3   120k, 75k G(A)      60-120        +                                     5B6   120k, 75k                 HSV-gD25                                                                              +                                     5B9   120k, 55k J(A/B/D)                +                                     5C2   120, 55, 35k                                                                            J(D)     390-439                                                                              421-432 +                                     5D6   120k, 55,                         +                                     5G9   120, 55, 35k                      N.D.                                  6D8   120k, 75k K         86-115        +                                     6E10  120k, 75k B         60-320        -                                     7F11  120, 55, 35k                                                                            J(D)     431-448                                                                              413-457 +                                     7G11  129, 55, 35k                                                                            J                       N.D.                                  9E3   160k      H(C)     542-573        +                                     9F6   120k, 75k L        240-275        +                                     10Cl  160k      D(E)                    +                                     10D8  120k, 75k A               301-324 +                                     10F6  120k, 75k A               301-324 +                                     11G5  120k, 75k A               301-324 +                                     13H8  120k, 75k F(D/E)   410-453                                                                              412-457 +                                     14F12 160k, 41k E(D/C)                  +                                     15G7  160k      C                       +                                     ______________________________________                                         *MAbs in italics were raised against gp120. All others were raised agains     683ΔC.7 (gp160).                                                   

To further explore the specificity of these antibodies, and to controlfor the possibility that the preferential reactivity to gp120 or gp160might represent an artifact peculiar to the ELISA assays, the binding ofthese antibodies was studied in a liquid phase radioimmunoprecipitationassay (RIP). For these experiments rgp120 and sgp160 were metabolicallylabeled with ³⁵ S!-methionine. Both proteins were then mixed togetherand reacted with the monoclonal antibodies. The resulting antibodyantigen complexes were then absorbed to glutaraldehyde fixed S. aureusand the proteins specifically immunoprecipitated were resolved bypolyacrylamide gel electrophoresis as previously described. FIG. 5 showsan autoradiograph where the ten monoclonal antibodies to sgp160 wereanalyzed. It can be seen that three major bands were specificallyimmunoprecipitated by one or more of the sera tested, a 140 kD bandcorresponding to sgp160, a 110-120 kD band corresponding to rgp120, anda 75 kD band which represents proteolytic breakdown products of gp120and sgp160. Previous studies, described above, have indicated that gp120and sgp160 produced in recombinant cell culture are often clippedbetween amino acid residues 315-316. In the case of gp120, proteolysisat this residue yields an amino-terminal 75 kD fragment andcarboxy-terminal 50 kD fragment which is in turn proteolyzed to smallerfragments. Proteolysis of sgp160 at this residue yields two fragmentswith a 75 kD mobility (gp75a and gp75b). Comparison of the data inTables 1 and 2 with that in FIG. 4 revealed that the RIP data correspondvery well with the ELISA data and that the monoclonal antibodies 9E3,10C1, 14F12, and 15G7 all were reactive exclusively with gp160. Thisresult suggests that those antibodies reacted with epitopes on gp41 orwith epitopes dependent on the interaction between gp41 and gp120. Thusit is concluded that the selectivity observed could not be attributed toan artifact in the ELISA format.

3. Function of MonoClonal Antibodies.

Six of the monoclonal antibodies inhibited the binding of gp120 to theCD4 receptor. Of these six, three were able to neutralize HIV virions asindicated by a reduction of reverse transcriptase activity in vitro.Three other monoclonal antibodies also neutralized infectious virions inthe in vitro reverse transcriptase assays. Assays were performedaccording to the following procedures.

1. CD4/gp120 binding: Monoclonal antibodies were tested in a solutionphase ELISA assay to determine CD4/gp120 blocking ability. ELISAmicrotiter plates were coated with antibody to CD4 (L104.5) at 1.0μg/ml. In a separate reaction plate, gp160 or 120 antibodies wereincubated with soluble rgp120 overnight at 4° C. Soluble CD4 was addedfor one hour at room temperature, and the resulting complex wastransferred to the L104.5-coated plate. Non-blocked CD4 was detectedwith a horseradish-peroxidase-conjugated anti-CD4 antibody (Leu3a-HRP).If the CD4/120 binding site was blocked by a gp160/120 antibody, solubleCD4 would bind to the plate and would be detected by the peroxidaseconjugate.

2. Syneytia Inhibition Assay. Monoelonal antibodies were tested fortheir ability to inhibit syncytia formation with HIV(IIIB) infectedcells according to known protocols.

3. Reverse Transcriptase Assay. Monoclonal antibodies were tested for invitro neutralization of HIV in reverse transcriptase (RT) assays. Inthis assay, various dilutions of antisera were incubated with a stocksolution of the IIIB isolate of HIV-1. The antibody-treated virus wasthen added to a culture of H9 cells as previously described in theliterature, and after seven days of culture the reversetranscriptase-specific incorporation of thyrmidine was measured. Of 10antibodies tested four were found to inhibit HIV-1 infectivity in thisassay. Greatly reduced levels of reverse transcriptase activity ininfected CD4+ T-lymphocyte cultures may indicate neutralization of thevirus by the monoclonal antibody. A positive result indicates greaterthan 50% RT inhibition at a minimum 1:10 dilution (approximately 50-100μg/ml).

Results of these assays are summarized in table 4.

                  TABLE 4*                                                        ______________________________________                                        MAb     CD4/gp120     Syncytia RT Assay                                       ______________________________________                                        1D10    --                                                                    1F9     --                                                                    5B3     Inhibits               Inhibits                                       5B6     --                                                                    5B9     --                                                                    5C2     Inhibits                                                              5D6     --                                                                    5G9     --                                                                    6D8     --                                                                    6E10    Inhibits               Inhibits                                       7F11    Inhibits                                                              7G11    Inhibits                                                              9E3     --                                                                    9F6     --                                                                    10Cl    --                                                                    10D8    --            Inhibits Inhibits                                       10F6    --            Inhibits Inhibits                                       11G5    --            Inhibits Inhibits                                       13H8    Inhibits               Inhibits                                       14F12   --                                                                    15G7    --                                                                    ______________________________________                                         *MAbs in italics were raised against gp120. All others were raised agains     683ΔC.7 (gp160).                                                   

4. Discussion.

Of the twenty-one monoclonal antibodies defining eleven differentepitopes of gp160, nine have identified functional epitopes of gp120based on the available assays. Six of these antibodies blocked thegp120/CD4 interaction. Of these six, three anti-rgp120 and oneanti-683ΔC7 MAb bound a region of gp120 that was defined as critical forCD4 binding (Lasky et el., supra). The two other blocking antibodies6E10 and 5B3, which were made against 683ΔC.7, appear to bind regionswhich are not in close sequential proximity to the earlier-reported CD4binding region. Whether they block sterically or through some otherinteraction is knot known. The interest in the 6E10 and 5B3 antibodiesis further enhanced by their ability to neutralize the virus asindicated in the reverse transcriptase assays. It is possible that theseantibodies will define other regions of gp120 critical for infectivity.

The 10D8, 11G5 and 10F6 antibodies, also raised against 683ΔC.7, bind a24 amino acid region (amino acids 301-324) designated as RP-135 byearlier published reports (Matsushita et al., J Virol. 62(6):2107-2114,1988). Data from the syncytia inhibition assay was consistent with otherreports of antibodies to the RP-135 region. Exactly how this region isinvolved in neutralization is not understood, however, it is known thatit lies outside the gp120/CD4 binding site. More importantly, thisregion spans the internal gp120 clip cite (at amino acids 315-316).These antibodies, or other antibodies to this region, are used toseparate out clipped gp120 when a preparation of unclipped gp120 isdesired.

Other monoclonal antibodies bound epitopes on gp120 that are highlyconserved among the different isolates, however no function has yet tobe assigned to these regions. Although some rgp120 and sgp160 MAbsappear to bind the same epitope, only those raised against sgp160demonstrate functional activity. This suggests that the 683ΔC.7 moleculemay preserve the conformational structure essential for the generationof neutralizing MAbs.

In our studies we explored the cross reactivity of antibodies elicitedagainst the IIIB isolate of HIV-1 with other isolates. Since thesequence of gp120 has been determined for a large number of HIV-1isolates, cross reactivity data might be useful in epitope mappingstudies. For these studies the env genes of five diverse isolates ofHIV-1 were mutagenized so as to insert a stop codon near the authenticterminus of gp120. To enhance expression in mammalian cells, the signalsequences were deleted and replaced with that of glycoprotein D ofherpes simplex virus type 1 as previously described. The reactivity ofthese isolates with the monoclonal antibodies to sgp160 was measured intwo ways: in a dot blot assay using partially purified envelopeglycoprotein coupled to nitrocellulose, and in aradioimmunoprecipitation assay.

To measure the reactivity of the monoclonal antibodies with authenticHIV-1 derived viral proteins, immunoblot strips purchased from Dupontwere used. In these studies 10 μl of ascites were diluted to 3 ml inblocking buffer and incubated with the nitrocellulose strips for 1 hourat room temperature. The strips were then washed three times with 3 mlof blocking buffer and then incubated with a 1:1000 dilution of alkalinephosphatase conjugated, affinity-purified, goat anti-mouseimmunoglobulin for 1 hour. The strips were then washed three times andstained with a phosphatase developing kit commercially available fromKirlegaar and Perry. Results of this procedure are shown in FIG. 4.

As can be seen in FIG. 4, monoclonals 13H8 and 5B3 were able to reactwith all of the isolates tested. Similar analysis was carried out on anumber of the monoclonal antibodies to gp120 previously described. Inthese studies another antibody, 6D8, was found to react with allisolates tested.

To explore the potential that these monoclonal antibodies could becoupled to cytotoxic agents and used as immunotoxin conjugates, theability of these antibodies to bind to env glycoproteins in the presenceof human sera that contains high levels of antibodies to gp120 wasmeasured. To ascertain whether or not monoclonal antibodies to gp120would be inhibited by the antibodies present in the sera of HIV-1infected individuals, competitive binding assays were performed. ELISAmicrotiter plates were coated with gp160 at 1 μg/ml, blocked and washed.Normal human sera was then added at 1:10 or 1:100 and an active fractionof sen from HIV-1 infected individuals was added at 100 μg/ml. Sera waspro-incubated for one hour, and then various concentrations of purifiedmonoclonal antibodies at 100 μg/ml. After a one-hour incubation, plateswere washed and antibody detected with GAM-HRP.

We found that the monoclonals were able to bind even in the presence ofundiluted human sera. This result was somewhat surprising and suggeststhat either the monoclonals were directed to epitopes different fromthose recognized by antibodies in human sen, or that the concentrationand/or affinity of a monoclonal antibody to any given epitope is muchhigher than the concentration and/or affinity of antibodies to anyparticular epitope in human polyclonal sofa. Thus these monoclonalscould be passively transferred and are expected to bind to HIV-1glycoproteins in the presence of high concentrations of human sera.

Example 3 PREPARATION OF UNCLIPPED GP120 THROUGH RECOMBINANT CELLCULTURE

Recombinant cell culture procedures have been developed which yieldunclipped HIV env polypeptides (in this example rgp120) which aresubstantially free of clipped gp120 fragments. Current approaches tocell culture optimization are found in Mather, Methods in Enzymology,vol. 185, chapter 43, pages 567-577 (1990), specifically incorporated byreference.

Chinese hamster ovary cells transfected with an HIV envelope geneexpression plasmid (CHO DHFR+) (Lasky, L. A. et al., Science 223:209;1986) were used for the production of the rgp120 used in this example.An initial stock spinner culture was initiated at approximately 3.11×10⁵cells/ml and grown in 1.5 liters of selective medium containing lowglucose (3.2 g/l), 7.5% diafiltered fetal bovine serum, and 3.7 ml of1.0 mM methotrexate. The culture was stored on magnetic stirrer in a 37°C. (30°-41° C.) incubator at approximately 40-50 rpm. After four days,the culture reached a density of approximately 15.0×10⁵ cells/ml(approximately 98% viability). Approximately 800 ml of this stockspinner culture was placed in roller bottles containing about 7200 ml ofselective medium as before. Roller bottles were placed on roller racksat speed of approximately 0.3 rpm, at 37° C. (36°-40° C.) for four days.Dulbecco's modified Eagle's medium or Ham's F12/DME GHT⁻ medium issuitable, as are commercially available media designed for the growth ofcells frequently used for expression of recombinant proteins such as CHO(e.g., Hana Biologicals CHO medium).

After four days, the selective medium was poured out of the rollerbottles, and the bottles were rinsed and then filled with approximately300 ml of non-selective medium containing high glucose (4.3 g/l),methotrexate as above, and 1% fetal bovine serum. It is currentlypreferred that this stage of the cell culture be performed in low serum,preferably in media containing approximately 0-3 percent serum, and morepreferably 1 percent. The roller bottles were gassed with 100% CO₂ at2-4 psi for approximately 1-3 seconds through sterile pipers, and thenplaced on roller racks at approximately 0.3 rpm and 37° C. (36°-40° C.).

Cultures were harvested after approximately three days. To harvest, themedia in the roller bottles was poured out and filtered through anautoclaved 5.0 micron Millipore and 0.45 micron Sartorions filter, 0.2g/l sodium azide was added, and the harvest was stored at 2°-8° C. Anadditional 300 ml of non-selective media was to the roller bottles, andthe cultures were incubated as before for an additional three days andthen harvested a second time as before.

Harvested materials were concentrated by ultrafiltration, then purifiedusing affinity chromatography as described in Lasky et al., supra 1986.Additional purification procedures were performed, including gelpermeation chromatography and anion exchange chromatography, however itwas preferred to utilize cation exchange and hydrophobic interactionchromatography (HIC) according to standard protocols as described above.

Purification procedures utilizing the monoclonal antibodies disclosedabove, particularly 10F6, 6E10, 10D8 and 11G5, coupled toglycerol-coated controlled pore glass were also performed usingpublished procedures.

We claim:
 1. A method for recovering unclipped HIV env which is greaterthan 50% free of clipped HIV env comprising the following steps:a.contacting a first preparation of HIV env with an antibody directed toan HIV env epitope spanning the clip site for a time sufficient topermit formation of a second, antibody-bound unclipped HIV envpreparation; b. separating the second preparation from any HIV env whichis not antibody-bound; and c. recovering the unclipped HIV env from saidsecond preparation.
 2. A method for the isolation of unclipped HIV envwhich is greater than 50% free of clipped HIV env, comprising affinitychromatography wherein antibody directed to an HIV env epitope spanningthe clip site is bound to a carrier matrix and a solution containing HIVenv and unclipped HIV env is passed over the column and unclipped HIVenv is selectively adsorbed to the matrix-bound antibody, the adsorbedantibody-unclipped HIV env matrix is washed to remove non-adsorbedmaterial, and the unclipped HIV env is eluted.
 3. The method of claim 1wherein the unclipped HIV env is at least 90% free of clipped HIV env.4. The method of claim 1 wherein the antibody is selected from the groupof antibodies produced from hybridoma cells having ATCC DepositDesignations CRL 10511, CRL 10512, and CRL
 10513. 5. The method of claim2, wherein the unclipped HIV env is at least 90% free of clipped HIVenv.
 6. The method of claim 2, wherein the antibody is selected from thegroup of antibodies produced from hybridoma cells having ATCC DepositDesignations CRL 10511, CRL 10512, and CRL 10513.